Tatsumi Adachi

Influence of LIF and BMP-2 on differentiation and development of glial cells in primary cultures of embryonic rat cerebral hemisphere.

Cells prepared from the cerebral hemisphere of embryonic Day 18 rats were maintained for 2 days in serum‐free modified Bottenstein‐Sato (mBS) medium containing thyroid hormone (TH), with or without leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP)‐2, and these influences on the differentiation and development of glial cells were investigated using the cells maintained in mBS medium containing TH as controls. The levels of glial fibrillary acidic protein (GFAP) expression and the number of GFAP‐positive astrocytes increased markedly with the addition of LIF or BMP‐2, and were enhanced further with the addition of both LIF and BMP‐2. The number of O1‐positive oligodendrocytes increased with the addition of LIF, whereas it decreased with the addition of BMP‐2. The number did not change with the addition of both cytokines. Using antibody against platelet‐derived growth factor (PDGF), we then excluded indirect effects of these cytokines through PDGF, which would increase by accelerated astrocyte development. When PDGF was neutralized, the number of oligodendrocytes increased under all conditions examined. As a result of the neutralization, the effect of BMP‐2 on oligodendrocyte differentiation was eliminated, although LIF remained effective. These results suggest that the differentiation of oligodendrocytes was delayed partially by PDGF even in control cultures. It is also suggested that LIF and BMP‐2, each of which accelerates the differentiation and development of astrocytes, would seem to have different effects on oligodendrocyte differentiation, i.e., LIF would directly affect oligodendrocyte differentiation, whereas BMP‐2 would affect it mainly through PDGF.

Inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cAMP-induced differentiation of rat C6 glial cell line

Dioxin is suspected to cause adverse effects on the development of the central nervous system (CNS). To investigate the neurotoxic effects of dioxin on the differentiation of astrocytes, rat C6 glial cell line was used as a model, because these cells are induced to express astrocyte markers and to change the cell morphology toward an astrocytic phenotype by increasing intracellular cAMP levels. When C6 cells were simultaneously exposed to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) and N6,O2′‐dibutylyl cAMP (dbcAMP), the expression of cytochrome P‐450 1A1 (CYP1A1) was dramatically increased, and the expression of aryl hydrocarbon receptor (AhR) was moderately decreased in a dose‐dependent manner. In addition, extension of astrocytic processes was inhibited by 1 nM TCDD that did not reduce cell viability. TCDD also inhibited the induction of glial fibrillary acidic protein (GFAP) expression in a dose‐dependent manner, until the end of a 72‐hr exposure period. This inhibition was restored by the addition of an antagonist of AhR, α‐naphthoflavone. These results indicate that TCDD inhibits astrocytic differentiation of C6 cells, which may be mediated by an AhR‐dependent pathway. J. Neurosci. Res. 64:402–409, 2001.

Selective disappearance of an axonal protein, 440‐kDa ankyrinB, associated with neuronal degeneration induced by methylmercury

The 440‐kDa isoform of brain ankyrin, 440‐kDa ankyrinB, is a neuron‐specific protein and is confined to axons. Cerebellum is one of the areas characteristically altered by methylmercury intoxication both in the adult and during development. When rat cerebellar neurons matured for 7 days in vitro were exposed to methylmercury at 0.03 μM for 48 hr, viability of the cells was unaffected. However, the immunocytochemical staining of 440‐kDa ankyrinB diminished drastically, whereas that of microtubule‐associated protein‐2, which is localized in dendrites and cell bodies, and of glial fibrillary acidic protein (GFAP), a marker for astroglial cells coexisting in the culture, remained unchanged. To confirm these observations, a simplified dot blot assay was established to determine 440‐kDa ankyrinB and several other marker proteins in cultured cell samples. With this assay, we found that methylmercury at a submicromolar range induced a decrease of 440‐kDa ankyrinB and an increase of GFAP in a dose‐dependent manner in cerebellar cells in primary culture. Surprisingly, another axonal protein, tau, remained mostly in its intact molecular sizes even in the presence of 0.3–1.0 μM methylmercury, though its immunocytochemical localization was substantially altered. These results indicate that selective loss of the axonal protein 440‐kDa ankyrinB is associated with the early stage of degeneration of cerebellar neurons induced by methylmercury. Therefore, 440‐kDa ankyrinB should be useful as a specific and sensitive marker for the neurotoxicity of methylmercury.

Neutral glycolipid and ganglioside composition of type‐1 and type‐2 astrocytes from rat cerebral hemisphere

We reported previously that the major gangliosides in primary mixed‐type astrocyte cultures are GM3 and GD3. To obtain more information regarding the exact distribution of glycosphingolipids in different types of astrocytes, we established a line of type‐1 astrocytes that are characterized by a Ran‐2 positive, broad flat morphology, and by the absence of binding to A2B5 antibodies. We also purified O‐2A progenitor cells by immunopanning and cultured them in the presence of 10% newborn calf serum. They differentiated into type‐2 astrocytes that were identified by immunostaining for each of GD3, A2B5, and GFAP. Using these cell cultures, we demonstrate that the major gangliosides were GM3 in type‐1 astrocytes and GM3 and GD3 in type‐2 astrocytes. In addition, a set of neutral glycolipids was identified based on the HP‐TLC migration properties of CMH, CDH, CTH, and Glob, but the component distribution of these glycolipids is related to that of glycolipids of astrocytes. A marked increase in the expression of CTH and Glob was shown in type‐2 astrocytes. The amount of neutral glycolipid‐sugar was higher in the type‐2 astrocytes than in the type‐1 astrocytes. These results suggest that the increase in the total glycosphingolipid content and the change in the neutral glycolipid composition produced by type‐2 astrocytes may be related to their biological functions and the cellular compositions. J. Neurosci. Res. 55:382–393, 1999.u2003

Influence of cell density and thyroid hormone on glial cell development in primary cultures of embryonic rat cerebral hemisphere.

The influence of cell density and thyroid hormone (TH) on the development of astrocytes and oligodendrocytes was investigated in primary cultures prepared from rat cerebral hemisphere on embryonic day (E)18. At the beginning of the culture, most of the cells were microtubule‐associated protein 2 (MAP2)‐positive neurons, whereas O1‐positive oligodendrocytes and glial fibrillary acidic protein (GFAP)‐positive astrocytes were rarely observed. After the cells were maintained in serum‐free defined medium, astrocytes developed at high cell density but rarely at a low one. When leukemia inhibitory factor (LIF) was supplemented in low‐density cultures, the levels of GFAP expression markedly increased to almost the same extent as in high‐density culture without TH. This suggests that, in low‐density cultures, astrocyte progenitors could not differentiate because of insufficient astrocyte‐inducing factors. Interestingly, the addition of TH increased GFAP expression levels only at high density. The number of oligodendrocytes increased with TH addition at both cell densities, although the effects were more remarkable at high density. These results suggest that cell density and TH are pivotal factors in the development of both astrocytes and oligodendrocytes. It is also suggested that the effects of TH on glial cell development could be accelerated via cell–cell communications.

Expression and localization of brain ankyrin isoforms and related proteins during early developmental stages of rat nervous system.

Abstract: Expression and localization of two isoforms of brain ankyrin, 440‐ and 220‐kDa ankyrinB, were studied in the developing nervous system of the rat fetus. The 440‐kDa ankyrinB appeared on as early as embryonic day 13, and its level increased progressively toward the day of birth, which was similar to the expression pattern of growth‐associated protein (GAP)‐43, a well‐established axonal protein. On the other hand, 220‐kDa ankyrinB was expressed at a low level but constitutively throughout the latter prenatal period and was a major isoform even before embryonic day 14. Whereas the localization of 440‐kDa ankyrinB was essentially confined to the axons, judging from the similarity with that of GAP‐43, 220‐kDa ankyrinB showed a rather general distribution in neural tissue. The localization of L1, known as an ankyrinB‐binding protein, was similar to that of 440‐kDa ankyrinB in the brain tissue, whereas it was similar to that of 220‐kDa ankyrinB in cultured neurons, suggesting that the interaction of L1 with brain ankyrins in neurons is affected by their environment.