Thyroidal influence on the cell surface GM 1 of granule cells: Its significance in cell migration during rat brain development

Cellular and Molecular Neurobiology, Vol. 12, No. 6, 1992

Thyroidal Influence on the Cell Surface GM1 of Granule Cells: Its Significance in Cell Migration During Rat Brain Development

Munmun Chakraborty, ~ Pulak Lahiri, 2 and Diptendu Chatterjee ~'a

Received Jane 30, 1991; accepted April 23, 1992

KEY WORDS: granule cells; GM1 ganglioside; thyroid hormones; cell migration.

SUMMARY

1. No difference was observed in the in vitro growing ability of granule cells isolated from hypothyroid or normal rat brain. When granule cells were taken from hypothyroid rat brain and grown in normal culture medium containing 10% fetal calf serum, they behaved similarly to the granule cells obtained from normal rat brain.

2. In both cases there were progressive losses of in vitro growing ability of the granule cells with the age of the animal and it became impossible to grow them when derived from 21 days or older animals.

3. A marked decrease in cell surface GM1 was observed when the cells were maintained under thyroid hormone-deficient conditions in culture.

4. Anti-GM1 antibody was found to inhibit significantly the migration of granule cells along the astrocyte fibers.

5. These results indicate that GM1 has an important role in thyroid hormone-dependent postnatal brain maturation in rat.

INTRODUCTION

Thyroid hormone status has a profound influence on neural development during the first few weeks after birth in rat brain. Proper level of thyroid hormone is

1 Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06511.

2 Department of Zoology, University of Calcutta, 35 Ballygunge Circular Road, Calcutta--700 019, India.

3 To whom correspondence should be addressed.

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0272-4340/92/1200-0589506.50/0 © 1992 Plenum Publishing Corporation

590 Chakraborty, Lahiri, and Chatteriee

essential for normal rate of germinal cell proliferation, cessation of cell division, formation of neurons from precursor cells, axonal and dendritic outgrowth, migration of granule cells, and formation of correct number and types of synaptic junctions (Clos and Legrand, 1973; Bass et al., 1977; Lauder, 1979). The defects of hypothyroidism can be recovered by thyroid hormone therapy if it is given early enough (Rabie and Legrand, 1973). One of the major effects of hypothyro- idism is on the granule cells of the cerebellum (Lewis et al., 1976). Very little is known about the difference between the granule cells of normal brain and those of hypothyroid rat brain. Balazs et al. (1985) concluded, on the basis of their studies on some parameters, v i z . , protein content, development of D2 protein, voltage-sensitive 22Na uptake, and muscarinic receptor content, that thyroid hormones do not affect granule cells directly--the defects which were observed in granule cells under hypothyroid conditions were secondary to the effects on Purkinji cells.

In this paper we demonstrate that when granule cells were maintained under thyroid-deficient conditions in vi tro for 2 weeks, the cells acquired about 61 + 9% less GM1 gangliosides on their surface compared to cells maintained in the presence of thyroid hormones. We have also demonstrated, using anti-GM~ antibody (Fab fragment), that this reduced cell surface GM1 content might be partially responsible for the failure of granular cells to migrate during hypothyroidism.

M A T E R I A L S A N D M E T H O D S

Cerebellar granule cells were cultured according to Fields et al. (1982). Briefly, cerebella of rats of the indicated ages were dissected, meninges were removed and chopped, and pooled cerebella were digested with trypsin and dissociated by trituration in the presence of albumin, soybean trypsin inhibitor, and DNAse 1. Dead cells and debris were removed by centrifugation of the cells through a 4% albumin solution, and 0.3 to 0.5 × 106 cells were plated onto poly-L-lysine-coated glass coverslips in Eagle's minimal essential medium plus 10% fetal calf serum, 0.6% glucose, 25 mM KCI, and 5/~g/ml of gentamycin. Flurodeoxy Uridine (FUdR) (8 × 10 -5 M) and uridine (35 ~g/ml) were added to the culture at 24 hr and half the medium was changed twice per week with the FUdR-uridine containing medium. Poly-L-lysine (PLL), high glucose, and high KCI were required for maximum cell viability and slower cell aggregation. In another set of experiments granule cells were grown in serum-free medium according to Ahmed et al. (1983) in the presence or absence of 0.45 nM triiodothyronine (T3).

Rats were made hypothyroid by force-feeding of propylthiouracil as de- scribed by Mazumdar et al. (1985). Anti-GMl antibody was developed in rabbits according to Naiki et al. (1974). The Fab fragment from the IgG fraction (purified by protein A-agarose affinity column) were prepared according to Hudson and Hay (1976). The Fab fraction was affinity purified from GMl-containing lipo- somes according to Naiki et al. (1974). The affinity-purified antibody has little cross-reactivity toward GDlb compared to that against GM1.

Thyroidal Influence on GM1 of Granule Cells 591

For radioimmunoassay of GM1, cells were grown on PLL-caoted coverslips and reacted with cholera toxin (1/~g/ml) after blocking nonspecific binding with egg albumin (10 mg/ml). After wash, the cells were incubated with anticholera toxin (developed in our laboratory in rabbits) followed by 125I-labeled anti-rabbit IgG. The bound radioactivity was counted after thorough washing.

Immunofluoresence staining with Ran-2 antibody (Barlett et al . , 1981) was carried out as described before (Chatterjee and Sarkar, 1987).

R E S U L T S A N D D I S C U S S I O N

Cerebellar granule cells grew well when isolated from the hypothyroid as well as normal 1-day-old rat brain; 74 + 6% of the plated cells were viable and there were long processes from each and every cell (Figs. 1A and 2A). In both cases, during the second week of culture the granule cells migrated along the astrocyte fibers (Fig. 3) and formed clusters of neurons and nerve fiber bundles. The cells could be grown for up to 40 days without any problem but there was progressively more clumping of the cells with the age of the culture. Cells from cerebella of 7-day-old hypothyroid and normal rats grew similarly, although there was slightly more loss of plated cells in both cases compared to that found in the case of 1-day-old rat brain (62 + 4% viable; Figs. 1B and 2B). These cells also could be grown for up to 40 days in v i tro. On the other hand, 72 :E 8% of the cells died when we plated cells from 14-day-old rat cerebel la--hypothyroid as well as

Fig. 1. Microphotographs of granule cells grown 10 days in vitro in the presence of serum after isolation from cerebellum of hypothyroid rats (A) 1 day, (B) 7 days (C) 14 days and (D) 21 days old. Plated cell densities were 0.3 x 106 cells/coverslip, x400; reduced 55% for reproduction.

592 Chakraborty, Lahiri, and Chatterjee

Fig. 2. Microphotographs of granule cells grown 10 days in vitro in the presence of serum after isolation from cerebellum of normal rats (A) 1 day, (B) 7 days, (C) 14 days, and (D) 21 days old. Plated cell densities were 0.3 × 106 cells per coverlsip. ×400; reduced 55%m for reproduction.

normal. But still the rest ( -30%) of the plated cells from 14-day-old rat cerebellum differentiated full in culture (Figs. 1C and 2C). Growth of the granule cells became impossible when the cells were derived from 21-day or older hypothyroid as well as normal rat cerebellum. There was initial attachment of 65 + 9% of the cells but they came out of the plates within 3 days without forming any processes (Figs. 1D and 2D).

The in vitro growth of granule cells isolated from hypothyroid rat cerebellum

Fig. 3. Microphotographs of a cerebellar granule cell culture (7 days) showing the arrangements of neurons along the astrocyte fibers. (A) Phase-contrast microphotograph showing the neurons. (B) Immunofluorescence staining of the same field with Ran-2 antibody, a cell surface marker for astrocytes showing the astrocyte present underneath the neurons. Cells were fixed with 1% acetic acid in ethanol, mounted, and observed at a 1000x magnification. Reduced 40% for reproduction.

Thyroidal Influence on GM1 of Granule Cells 593

and maintained in the presence of the thyroid hormone (present in the serum used in the medium) was found to be similar to that observed in the case of granular cells isolated from normal rat cerebellum. But when the granule cells derived from normal or hypothyroid newborn rat cerebella and maintained under thyroid-deficient conditions in serum-free medium, partial loss of the granular cell migration along the astrocytes fibres and cell cluster formation were observed. About 42 + 6% of the cells remained isolated in the absence of T3 compared to that observed in the presence of T3 (16 + 6%) in serum-free medium (Figs. 4A and B). This type of blocking of cell-cell adhesion in the absence of thyroid hormones in culture was also reported by Balazs et al. (1985), but they did not observe any influence of thyroid hormones on this cell-cell aggregation in 7 days. We also did not observe any major affect of thyroid hormones in the first week of culture, but effects were observed in the second week.

It is well documented that GM1 gangliosides has pronounced effects on neuronal cell growth (Leon et al., 1982; Roisen et al., 1981; Toffano et al., 1983). Nerve growth factor (NGF)- or condition medium mediated neurite outgrowth of dorsal root ganglionic neurons was reported to be inhibited by poly- or monoclonal antibody against GM1 (Schwartz and Spirman, 1982; Spoerri et al., 1988). Greis and Rosner (1990) reported that monoclonal antibody against c-polysialo gangliosides could inhibit migration of neurons in primary cultures of embryonic chicken optic lobe. As granule cells acquire more and more GM~ on their surface during their migration from the upper granular layer to the inner layer (Willinger and Schachner, 1980) during the period of thyroid hormone- dependent maturation of rat brain (Chatterjee and Sarkar, 1987), we studied the influence of thyroid hormones on the GM~ content of the granule cell surface in vitro. It was observed that when the cells were grown in serum-free medium, the GM1 content was about 50-70% greater in the presence of T3 compared to that observed in the absence of T3 (Fig. 5). It is interesting that the GM~ content of the cells increased rapidly, within 6 days in vitro, and then reached almost a steady state. It was reported by Vitiello et al. (1989) that in the presence of thyroid hormones, though there was little change in the molar percentage of different gangliosides, total cerebellar gangliosides per milligram of DNA (or per

Fig. 4. Microphotographs of cerebellar granule cells grown 10 days in vitro in serum-free medium (A) in the presence and (B) in the absence of 0.45 nM T 3. Plated cell densities were 0.3 x 106 cells/coverslip. ×400; reduced 55% for reproduction.

594 Chakraborty, Lahiri, and Chatterjee

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Fig. 5. Radioimmunoassay of the cell surface GM1 content of granule cells isolated from newborn rat cerebellum and grown in serum-free medium in the presence (A) or absence (B) of 0.45 nM T 3.

milligram of protein) were increased about 50% over the level observed under thyroid-deficient conditions. This inidcated increase in the level of all the individual gangliosides per cell as well as per milligram of protein in the presence of thyroid hormones---which is in good agreement with what we observed for the GM1 content of cerebellar granular cells the presence of the thyroid hormones.

To understand whether this low level of granule cell surface GM1 under thyroid hormone-deficient conditions is responsible for the failure of granule cell migration, we have used immunological approaches with anti-GM1 antibody (purified Fab fragment). When the granule cells were incubated with the purified Fab fragment of anti-GM1 antibody at a 4 /~g/ml concentration, no significant effect on neurite outgrowth was observed. The effect of the antibody at this concentration was observed in the second week of culture---the normal migration

Fig. 6. Effects of afffinity-purified anti-GM1 antibody (Fab fragment) on the migration of granule cells in culture. Granule cells from rat cerebellum were grown in the presence of normal mouse IgG for 16 days (A) and in the presence of the Fab fragment of anti-GM l antibody (affinity purified) for 16 days (B). Note the greater clumping of the cells in 16-day than in 10-day culture (A). An antibody against minor brain gangliosides, A2B5, had no effect on cell migration or cell growth (data not shown). ×400; reduced 55% for reproduction.

Thyroidal Influence on GM~ of Granule Cells 595

of the granular cells along the as t rocyte fibers was d e l a y e d - - t h o u g h not completely inhibited. E v e n after 16 days in v i t ro , about 70 + 8% of the cells remained isolated in the presence of ant ibody, c o m p a r e d to only 14 + 4% isolated cells in the presence of normal rabbit IgC at the same dilution (Figs. 6 A and B). We have also used the Fab f ragment f rom another ganglioside ant ibody, A 2 B 5 (which binds to minor gangliosides of cerebel lar neurons (F redman et al . , 1984), but did not observe any significant inhibition of cell g rowth or cell migrat ion.

A l though our unders tanding of the molecular mechan i sm of granule cell migrat ion is far f rom comple te and much remains to be learned, it is wel l -known that a p rominen t fea ture during the deve lopment of the nervous system is the order ly fo rmat ion of hor izontal cellular and fibrous layers. This morphogen ic process depends heavily on o rde red cell migra t ion and complex interact ions between neurons and glial cells (Rakic, 1971). Thyro id h o r m o n e was found to be essential for cell migrat ion during matura t ion o f cerebel lum (Grave , 1977). The results p resented here indicate that, in the absence of thyroid h o r m o n e , granular cells express lower level gangliosides on their surface. Significant inhibit ion of cell migrat ion by anti-GM1 ant ibody indicates that cell surface G M t might play a critical role in thyroid h o r m o n e - d e p e n d e n t cerebel lar granule cell migra t ion and cell-cel l contact during brain deve lopment .

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