APOPTOSIS IN THE DEVELOPING CEREBELLUM OF THE THYROID HORMONE DEFICIENT RAT

Qianxun Xiao and Vera M. Nikodem

Mechanism of Gene Regulation Section, Genetics and Biochemistry Branch, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 10 Center Dr. MSC 1766, Bethesda, MD 20892-1766

Received 8/20/98 Accepted 9/7/98

5. DISCUSSION

Although it is not arguable that TH is crucial for the cerebellum development, it has not been clearly established whether the arrested development of Purkinje cells dendrites, the primary synaptic target of granular cells, in the hormone deficient cerebellum, leads to the increased apoptotic activity of granular cells or whether the reduced number of granular cells connections with Purkinje cells affects Purkinje cell maturation. Indeed, the molecular basis for TH during brain development are poorly understood. Hormone action is mediated by thyroid hormone receptors especially the ß1, whose level reaches its highest during the first 2-3 weeks after birth. Thus, at the level of transcription, TH can regulate expression of variety of factors known to play an important role in the regulation of neuronal survival during development (28,29,30,31). Lindholm et al (32) reported that expression of neurotrophin 3 is under control of TH. Recently, Neveu et al (27)showed that neurotrophin 3 and brain derived neurotrophin factor prevent induced cell death of granular cells in hypothyroid cerebellum in vivo. Muller et al (33) reported that thyroid hormone also promotes expression of the bcl-2 protooncogene, the programmed cell death suppressor, and prevents apoptosis of early differentiating cerebellar granular neuron in vitro.

It is well established that programmed cell death plays an important role during maturation of the center nervous system. In this study, we have chosen to investigate the effect of the TH on the development of rat cerebellum since its maturation takes place postnatally. We have shown that the lack of TH increases and extends the apoptosis in the developing cerebellum and affects disappearance of the EGL.

In the rat, the cerebellum granular cells, a source of major excitatory afferents to the Purkinje cells, are generated from EGL (34). From p3 to p30, granular cells migrate to the IGL, by translocation of the cell body through the descending portion of the growing parallel fiber, leaving their axons (parallel fibers) in the ML forming synapse with the dendrites of Purkinje cells (21). Thus, it seems likely that growth of the descending portion of the parallel fiber is an important and perhaps rate-limiting aspect of granular cell migration and cerebellum maturation. It has been shown that the proper levels of TH are necessary to secure the growth of parallel fibers (35,36). Furthermore, hypothyroid rats show retardation in the proliferation, migration, and differentiation of cerebellar granular cells. Although a number of Purkinje cells do not appear to be affected by the lack of the hormone, there is, however, deficiency in the elaboration of Purkinje cell dendrite trees, spines and the synapses (18).

Using a sensitive method for detecting DNA fragmentation, a hall marker of apoptotic activity, our data showed that no apoptotic cells were detected in the euthyroid cerebellum at p2, 22 and p42. In agreement with previous results reported by Wood et al (37), the apoptosis reach its maximal value at p7 and then declined. It is during this period that most of the histogenesis of the cerebellum occurs. Thus, granular cell death may be a key factor in regulating the final number of neurons. Indeed, in the stage where the maximal rate of migration is finished, the apoptotic activity is descended and becomes undetectable at p22. This developmental stage is also marked by the absence of the EGL. In both the euthyroid and hypothyroid rats, the p8 apoptosis reached the peak level, however, there was a greater proportion of apoptotic cells in the IGL of hypothyroid rats than that in the control. Surprisingly, the granular cells were still undergoing apoptosis at p 22 in the IGL hypothyroid cerebellum, contrary to the control. Thus, it appears that the deficiency in TH during postnatal cerebellum development promotes and also extends the event of apoptosis. The presence of the EGL in hypothyroid cerebellum at p 42, the time when brain maturation should be completed, indicates that the hormone also plays a role in morphological changes accompanying brain development. Another striking morphological change in hypothyroid cerebellum is the impaired dendritic arborization of Purkinje cell in ML. Previous results showed that in hypothyroidism, retardation in the morphological maturation of Purkinje cells is the most apparent during the second postnatal week (22,38). We have shown that this period coincides with the peak of aggressive apoptosis augmented by the absence of the hormone. It is now important to uncover the mechanism whereby TH is able to control the cell cycle so as to limit the extent and time of apoptosis in the developing cerebellum.