Ana Maria Lennon

Rat, mouse, and guinea pig brain development and microtubule assembly.

The development of in vitro microtubule assembly and of tubulin concentration have been studied during brain maturation in the mouse and the rat, two species which have postnatal brain development, and in one species which is mature at birth, the guinea pig. (a) The rate of tubulin assembly is very slow soon after birth in both the mouse and rat; it increases progressively with age until adulthood. In contrast, in the guinea pig this rate is maximal at birth and slower rates are seen only at foetal stages. (b) Postnatal changes in the lag period of assembly and in the minimal concentration of tubulin (Cc) required to obtain in vitro assembly are seen in the mouse and the rat; in contrast these parameters are constant at all postnatal stages in the guinea pig with longer lag periods and lower Cc values being seen only at foetal stages. (c) Maximal rates of assembly, minimal lag periods, and minimal Cc values are restored after addition of microtubule‐associated proteins to foetal guinea pig or young mouse and rat preparations, suggesting that the difference in the kinetic parameters of assembly between these species depends on differences in the concentration or activity of these proteins. (d) Maximal tubulin concentrations are observed before birth in the guinea pig and approximately at day 10 in the rat and mouse. Lennon A. M. et al. Rat, mouse, and guinea pig brain development and microtubule assembly. J. Neurochem.35, 804–813 (1980).

Initiation of neurotubulin polymerisation and rat brain development.

In previous publications [ 1,2] we have shown that the concentration of tubulin is probably not the rate limiting factor for the assembly of neurotubules during rat brain development. Various data suggest that most, if not all, neurotubulin which is formed during an early stage of development (15 days of gestation) do not polymerize efficiently. The assumption was made that some factor, different from neurotubulin, is responsible for the initiation of the polymerization process. It was also shown that this initiator is probably the limiting factor for microtubule formation at early stages of development. Recent publications by Kirschner et al. [3] have reported that adult 6 S neurotubulin is unable to polymerize by itself unless another protein, the r factor, which acts as an initiator, is present. In order to determine whether this factor was responsible for the failure of the neurotubulin present in the brain supernatant of new born rats to polymerize normally, the effect of purified preparations of r was studied in this work. From different experiments it may be concluded that in early stages of brain development r, an ‘initiator’ of neurotubulin polymerization, is not present in sufficient amount to polymerize tubulin properly.

Cytosolic thyroxine-binding protein and brain development.

The properties of cytosolic thyroxine binding protein were studied in the cortex and cerebellum of the rat at different stages of postnatal development: (1) Polyacrylamide-gel electrophoretic analysis showed that rat-brain cortex and cerebellum contain the same cytosolic thyroxine-binding protein which is very similar to the liver-corresponding entity. No changes in the electrophoretic mobility were seen during development in the 2 brain regions. In contrast, no defined triiodothyronine-binding component could be observed by the same technique. (2) Kinetic analysis studies revealed that the equilibrium of binding is reached in approximately 10 min whatever the brain region, the concentration of cytosolic protein and the stage of development. In all these cases saturation was obtained with the same thyroxine concentration (approximately 5 x 10(-7) M). Scatchard analysis also showed that whatever the experimental conditions, brain cytosolic protein contains a single class of thyroxine-binding sites with a K A of approximately 8 x 10(7) M-1. (3) Comparison of the K A during development showed that this constant remains unchanged from day 3 after birth until day 35 in both the cortex and the cerebellum. In contrast the number of binding sites significantly decreases in the cortex (approximately 2-fold; p less than 0.001) from day 3 to 35 with an already significant decline from day 3 to 6 (p less than 0.001). In the cerebellum this decline was even more marked since almost no binding activity was left at adulthood. Comparison of cortex and cerebellum binding activities also showed that this latter region contains approximately half the binding sites (p less than 0.001) at every stage of development studied.

Cellular location of cytosolic triiodothyronine binding protein in primary cultures of fetal rat brain

The evolution of a cytosolic triiodothyronine (T3) binding protein was studied in primary cultures of fetal rat brain. These cultures exhibited neuronal characteristics during the first week. T3 binding activity in cell supernatants increased during this period from 39 +/- 7 (mean +/- SD) to 159 +/- 24 fmoles T3/culture flask. A similar increase was observed in the soluble proteins. After day 8, neuronal death occurred and glial cells multiplied and differentiated. On day 11 an 86% drop in the binding activity was observed (24 +/- 7 fmoles T3/culture flask); the pool of soluble proteins remained stable. Scatchard analysis revealed two types of binding site in both 7- and 14-day cultured cell cytosols. Binding affinities were similar in both cytosols (KA1 approximately 1.5 X 10(9) M-1, KA2 approximately 1 X 10(8) M-1); in contrast, the number of sites was 4-fold smaller in 14-day cytosols. In subcultures mostly composed of glial cells, almost the same affinities were measured, but the numbers of both types of sites were 20 times smaller than in 7-day cells. These results show that in cell cultures from embryonic rat telencephalon, cytosolic T3 binding protein is mainly located in the neurons.

A high affinity thyroid hormone binding protein in the cytosol of embryonic rat brain cells in primary cultures

A thyroid hormone binding protein(s) has been characterized in the cytosol of fetal rat brain cells in primary cultures. This protein is closely related to the one described in brain supernatants with respect to its electrophoretic mobility, binding kinetic parameters and estimated molecular weight (65 000 daltons). However, in contrast to the brain cytosolic binding protein, two classes of affinity sites for triiodothyronine (T3) and thyroxine (T4) have been demonstrated: a high affinity site (KA = 1.2-3.7(3) X 10(9) M-1 for T3 and KA = 3.7-5 X 10(8) M-1 for T4) and a low affinity site (KA = 0.8-1.4 X 10(8) M-1 for T3 and 1.6-2.9 X 10(7) M-1 for T4). The results are discussed with respect to their cellular significance.

12-O-Tetradecanoylphorbol 13-Acetate and Fibroblast Growth Factor Increase the 30-kDa Substrate Binding Subunit of Type II Deiodinase in Astrocytes

Abstract: Type II 5′‐deiodinase (D‐II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3′‐triiodothyronine (T3) in the brain., The D‐II activity in astroglial cell cultures is induced by several pathways including cyclic AMP (cAMP), 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), and fibroblast growth factors (FGFs). We have examined the effect of TPA and FGFs on the 30‐kDa substrate binding subunit of D‐II, by affinity labeling with N‐bromoacetyl‐[128I]T4 in astroglial cells. TPA (0.1 μM), 20 ng/ml acidic FGF (aFGF), and 1 mM 8‐bromo cyclic AMP all caused an increase in the 30‐kDa protein. cAMP induced the greatest increase (fivefold) followed by TPA (3.2‐fold) and FGF (2.8‐fold). Glucocorticoids acted synergistically with cAMP and aFGF and promoted the effect of TPA. Affinity labeling was competitively inhibited by bromoacetyl‐T4 > bromoacetyl‐T3 > T4 > reverse T3 > iopanoic acid > T3 > 3,5,3‐triiodothyroacetic acid. The effect of TPA (0.1 μM) was maximum at 8 h and then gradually decreased. aFGF (20 ng/ml) plus heparin (17 μg/ml) induced a maximal 30‐kDa increase at 8 h, which stayed stable for up to 24 h. The effect of aFGF was concentration dependent. Of the other growth factors studied, only basic FGF and platelet‐derived growth factor induced small increases in the 30‐kDa protein. Epidermal growth factor had little effect. In vitro labeling of cAMP, TPA, and aFGF‐stimulated cell sonicates resulted in an increase in the 30‐kDa protein that paralleled the increase in D‐II activity. These results correlate well with our previous studies showing that several distinct signaling pathways regulate D‐II activity. They suggest that the regulation of D‐II in astrocytes by cAMP, TPA, and aFGF involves an accumulation of the 30‐kDa substrate binding subunit.