In the present study, 3-day treatment of nondifferentiated NG108-15 cells with 100 nM angiotensin II (Ang II) induces morphological differentiation of neuronal cells characterized by the outgrowth of neurites. These morphological changes are correlated with an increase in the level of polymerized tubulin and in the level of the microtubule-associated protein, MAP2c. Mediation by the AT2 receptor may be inferred since: (a) these cells contain only AT2 receptors; (b) the effects are mimicked by CGP 42112 (an AT2 receptor agonist); (c) they are not suppressed by the addition of DUP 753 (an AT1 receptor antagonist); and (d) are abolished by co-incubation with PD 123319 (an AT2 receptor antagonist). Application of Ang II in dibutyryl cAMP-differentiated cells (which contain both types of receptors) induces neurite retraction, an effect mediated by the AT1 receptor. These results indicate that the AT2 receptor of Ang II induces neuronal differentiation, which is initiated through an increase in the levels of MAP2c associated with tubulin. Moreover, our results demonstrate that the AT1 receptor inhibit the process of differentiation induced by dibutyryl cAMP, whereas the AT2 receptors potentiate this effect, illustrating negative cross-talk interaction between the two types of Ang II receptors.

Phosphorylation of the head domains of intermediate filament proteins by second messenger-dependent kinases is important in regulating filament assembly. In the case of neurofilaments, head domain phosphorylation is known to be important in assembly, but few sites have been identified. Using matrix-assisted laser desorption-ionization (MALDI) and nano-electrospray mass spectrometry, we report the identification of several novel in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites in the low (NF-L) and middle (NF-M) molecular mass neurofilament subunits. Neurofilament polypeptides were purified from adult rat brain, and fractions containing a mixture of NF-L and NF-M were nonradioisotopically phosphorylated with PKA prior to proteolytic digestion of the polypeptides in situ in polyacrylamide excised from SDS gels. Sites of phosphorylation were determined by mass spectrometric analysis of mixtures enriched in tryptic phosphopeptides. In NF-L, four novel sites were identified: serines 12, 41, and 49 in the head domain and serine 435 in the carboxyl-terminal tail domain, and data consistent with phosphorylation of serine 2 were obtained. Recombinant rat NF-L protein was also phosphorylated with PKA, and the same serines were identified as phosphorylation sites, with two additional sites, serine 43 and probable phosphorylation of serine 55. In NF-M, one novel site, serine 1 in the amino-terminal head domain, was found to be phosphorylated, and serine 46, also in the amino-terminal head domain, was confirmed as a PKA phosphorylation site.

Microexplant cultures from three-day-old rats were used to investigate whether angiotensin II (Ang II), through its AT 1 and AT 2 receptors, could be involved in the morphological differentiation of cerebellar cells. Specific activation of the AT 2 receptor during 4-day treatment induced two major morphological changes. The first was characterized by increased elongation of neurites. The second change was cell migration from the edge of the microexplant toward the periphery. Western blot analyses and indirect immunofluorescence studies revealed an increase in the expression of neuron-specific ␤III-tubulin, as well as an increase in expression of the microtubule-associated proteins tau and MAP2. These effects were demonstrated by co-incubation of Ang II with 1 M DUP 753 (AT 1 receptor antagonist) or with 10 nM CGP 42112 (AT 2 receptor agonist) but abolished when Ang II was co-incubated with 1 M PD 123319 (AT 2 receptor antagonist), indicating that differentiation occurs through AT 2 receptor activation and that the AT 1 receptor inhibits the AT 2 effect. Taken together, these results demonstrate that Ang II is involved in cerebellum development for both neurite outgrowth and cell migration, two important processes in the organization of the various layers of the cerebellum.A large number of studies indicate that the hormone angiotensin II (Ang II) 1 and its receptors are present in the brain (1, 2). As in the periphery, the AT 1 receptor exhibits a high affinity for the nonpeptidic antagonist DUP 753 (Losartan), whereas the AT 2 receptor has a high affinity for the antagonist PD 123319 and the agonist CGP 42112 (1, 2). Although the AT 1 receptors are detected in areas involved in the regulation of blood pressure, hydromineral balance, and thirst, no central function has yet been attributed to the AT 2 receptor. This receptor is highly expressed in the inferior olive, locus coeruleus, thalamic nuclei, medial geniculate nuclei, and the molecular layer of the cerebellum (3-5).Although several studies have been conducted on the short term effect of AT 2 receptor activation on intracellular events, a few studies focused on the physiological function of the AT 2 receptor. One well described function is its antagonistic action on cellular growth induced by neurotrophic factors (nerve growth factor) (6, 7) or by the AT 1 receptor of Ang II (8 -10). Another function recently described for the AT 2 receptor is a role in programmed cell death (11,12). Interestingly, although the expression of the AT 1 receptor either remains stable or increases with development in rats, the expression and density of the AT 2 receptor decrease dramatically with maturation from fetal to neonatal to adult, both at the periphery and in several brain nuclei (13-16). This high and transient expression of the AT 2 receptor in fetal tissues suggests that it may play a specific role during development and cellular differentiation (7,11,17,18). Indeed, in a previous study, using neuroblastoma ϫ glioma hybrid NG108 -15 cells, we have shown that a 3-da...

The microtubule-associated protein, tau, is the principal component of paired helical filaments (PHFs) in Alzheimer's disease. PHF-tau is highly phosphorylated and a total of 25 sites of phosphorylation have so far been identified. Many of these sites are serine or threonine residues that are immediately followed in the sequence by proline residues, and hence are candidate phosphorylation sites for proline-directed kinases. In vitro, glycogen synthase kinase-3 (GSK-3), extracellular signal-related kinase-1 and -2, and mitogen-activated protein kinases, p38 kinase and c-jun N-terminal kinase, all phosphorylate many of these sites, although with different efficiencies for particular sites. Phosphorylation studies in transfected cells and neurons show that GSK-3 phosphorylates tau more extensively than do these other proline-directed kinases. Mutations in tau have been shown to affect in vitro phosphorylation of tau by GSK-3. The Arg406-->Trp (R406W) tau mutation also affects tau phosphorylation in cells.

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