J. Nunez

Developmental Expression of the Glial Fibrillary Acidic Protein mRNA in the Central Nervous System and in Cultured Astrocytes

Abstract: The expression of glial fibrillary acidic protein (GFAP)‐mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern‐blot evaluation using a specific cDNA probe, and cell‐free translation associated with immunoprecipitation. During brain maturation (4–56 days postnatal), the GFAP‐mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7–32 days in culture), the GFAP‐mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7–18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7–32 days), α‐tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP‐mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.

Effects of thyroid hormones during brain differentiation.

Since the placenta is practically impermeable to thyroid hormones (Fisher et al., 1977), the foetus depends for its brain development on the activity of its own thyroid gland: thyroid hormone deficiency at birth leads to severe physiological, biochemical and behavioural defects (Shapiro, 1977) which can be corrected only if an adequate replacement therapy is performed as early as possible. The most dramatic effects of thyroid hormones take place during the critical period of brain development, i.e. when the cell ceases to divide and begins to differentiate. Although several other abnormalities are observed in neonatal hypothyroidism (for instance defective myelination), probably the most significant of them is a hypoplastic neuropile, i.e. a reduction in electric activity of the brain and in the number of interactions between neurons (Eayrs, 1960; Legrand, 1967). A large variety of neuronal cell types is produced during brain differentiation and each mature neuron seems to be unique in its function. It therefore appears very difficult to expect that detailed studies will allow the description, in the near future, of how the differentiation programme allows the specification of the expression of each neuron. However, all neurons undergo a common major morphological transformation during brain differentiation, i.e. the formation of nerve cell processes which extend from the cell body to produce the dendrites and the axons. Such a dramatic change in cell shape is probably under thyroid hormone control and it seems to correlate well with the reduction in the number of connections seen in hypothyroidism. Changes in cell shape appear more and more directly related to both fast and slow modifications in the organisation of the cytoskeleton. This conclusion seems to apply not only to nerve cells but also to a variety of other cell types. In several of these systems thyroid hormones are required together with other factors to achieve complete maturation. Changes in the composition and organisation of the different elements which contribute to the building-up of the cytostructure are also good markers of neuronal cell differentiation, since neurite outgrowth not only implies large modifications in shape but also forward development of the growing processes. In this survey we shall successively and briefly describe (1) the aspects of the primary mechanism of action of thyroid hormones which have been found to be common to nerve cells and other cell types; (2) the different in vivo effects of thyroid hormone on the various parameters of brain development; (3) some of our recent attempts to ex

Immunological characterization of microtubule-associated proteins specific for the immature brain

G-i on a biochemical basis the effects of thyroid hormones on neurite outgrowth. No attempt has been made to refer to all articles published on these topics. A more detailed review is in press in the new edition of the Handbook of Neurochemistry (Nunez, 1984).

Microtubules and brain development

Immunoblotting analysis was used to detect the microtubule‐associated proteins present at different stages of rat brain development. Polyclonal antibodies were raised against the two main adult brain microtubule‐associated proteins: MAP‐2 (300 kDa) and TAU (60–70 kDa). Whatever the stage of development, anti‐MAP‐2 serum detected high molecular mass proteins and at immature stages a protein of 62 kDa. This protein which has previously been referred to as ‘young TAU slow’ is, therefore, immunologically related to MAP‐2. The anti‐TAU serum (but not the anti‐MAP‐2 serum) detected at immature stages of development a 48 kDa protein which also disappears at adulthood. This 48 kDa entity which has been referred to as ‘young TAU fast’ is progressively replaced by the closely spaced bands (60–70 kDa) of adult TAU proteins. The 62 and 48 kDa proteins appear therefore to be immunologically distinct and represent two microtubule‐associated proteins specific to the immature brain.

Regulation by thyroid hormone of microtubule assembly and neuronal differentiation

There has been considerable interest in the past few years in brain neurotubules: the concentration of microtubule protein is much higher in central nervous system cells than in other tissues [l] ; neurotubules are components of the axon and are believed to play an important role in axon function and in brain differentiation [2]. Thus neurotubules and neurotubulin might be good markers of axon differentiation. Bamburg et al. [3] using a time-decay colchicine binding assay and quantitative gel electrophoresis have found that microtubule brain protein concentration increases approximately two-fold between 5-7 days and 13 days of development in chick embryo brain, then returning slowly to the initial value in the adult. However these results do not provide an answer to the following questions: is neurotubulin progressively assembled to neurotubules as soon as synthesized? In this case the rate limiting factor would be the amount of tubulin present in the brain at each stage of development. Another possibility would be that all the neurotubulin would be formed before a given period of development without undergoing massive polymerisation, a specific signal inducing, at a precise stage of development, its polymerisation to neurotubules followed by rapid axon growth. An attempt is made here to answer these questions.

Expression of the mRNA for τ Proteins During Brain Development and in Cultured Neurons and Astroglial Cells

In this review we examine successively: 1) the major effects of thyroid hormone deficiency seen during brain development with special emphasis on the changes in neuronal morphology and migration occurring postnatally in the cerebellum. 2) The effects of this hormone on microtubule assembly during neurite outgrowth and acquisition of neuronal polarity. 3) The changes in expression of the different tubulin isoforms occurring during development in the normal and hypothyroid rat brain. 4) The regulation by thyroid hormone of the transition occurring during development between the juvenile and adult microtubule-associated protein Tau.

Regulation of the Glial Fibrillary Acidic Protein (GFAP) and of its Encoding mRNA in the Developing Brain and in Cultured Astrocytes

Abstract: Two τ cDNA probes of 1.6 and 0.3 kilobases (kb) have been used to study the expression of the τ mRNAs during mouse brain development and in highly homogeneous primary cultures of neurons and astrocytes. (1) Whatever the stage, a 6‐kb mRNA was detected with the two probes. In the astrocytes a 6‐kb mRNA hybridized clearly only with the 1.6‐kb probe. (2) During brain development the abundance of τ mRNA increases from a late fetal stage (— 4 days) until birth, remains high until 6 days postnatal, and then markedly decreases to reach very low values in adulthood. Such a marked decrease in the abundance of τ mRNA parallels that of α‐tubulin mRNA. These data suggest that: (1) depending on the stage of development and on the cell type (neurons or astrocytes) τ mRNAs of the same size encode several τ proteins differing in molecular weight: several τ proteins are expressed either during early stages of development (juvenile τ proteins of 48 kilodaltons) or in adulthood (mature τ proteins of 50–70 kilodaltons) or are specific of the astrocyte (83 kilodaltons). (2) The expression of the two major components of axonal microtubules, tubulin and τ proteins, seems to be developmentally coordinated.

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

The Glial Fibrillary Acidic Proctein (GFAP) is the monomer of a well characterized type of intermediary filaments, the fliofilaments, structurally identified as 10nm in diameter and which are essential components of the cytoskeletal architecture of the astrocyte (see Eng 1980 for a review). The expression of GFAP has been found to be highly specific of this cell type (Eng et al. 1971; Bignami et al. 1972; Uyeda et al. 1972; Gilden et al.1976: Ludwin et al. 1976; Ludwin et al. 1976; Lach and Weinmander 1978) and may therefore be used as an exclusive marker of astroglial cells.

EXPRESSION OF VARIOUS MICROTUBULE-ASSOCIATED PROTEIN 2 FORMS IN THE DEVELOPING MOUSE BRAIN AND IN CULTURED NEURONS AND ASTROCYTES

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).

Regulation of five tubulin isotypes by thyroid hormone during brain development

Abstract: A cDNA probe specific to microtubule‐associated protein 2 (MAP2) was used to study the expression of the mRNAs encoding the high‐ and low‐molecular‐weight MAP2 variants in cultured neurons and astrocytes. The timing and relative abundance of these MAP2 transcripts and of their encoded proteins were also studied in the developing cerebral hemispheres and cerebellum of the mouse. A 9‐kb mRNA, known to encode high‐molecular‐weight MAP2, was expressed in cultured astrocytes, albeit at a lower level than in neurons. The 6‐kb transcript, recently shown to encode low‐molecular‐weight MAP2 (MAP2c), was expressed in neurons and was the predominant MAP2 transcript of the astrocytes. The level of the 9‐ and 6‐kb transcripts decreased at late stages of astroglial and neuronal cell culture. The 9‐kb mRNA was detected in the cerebellum and cerebral hemispheres at every developmental stage. Although the levels of this mRNA varied slightly in the cerebral hemispheres, its expression was biphasic in the cerebellum. This might be explained by the differences in timing of development of the various neuronal cell types formed in these two brain areas. The 6‐kb transcript was detected only at early developmental stages in the two brain areas. Correlating the temporal expression of the 9‐kb mRNA to that of high‐molecular‐weight MAP2 indicates that the accumulation of this protein is in part regulated at a cytoplasmic level.