Filamentous tau pathology in nerve cells, astrocytes, and oligodendrocytes of aged baboons.

Intracellular filamentous inclusions containing abnormally phosphorylated tau protein are hallmarks of several human neurodegenerative disorders. This study reveals tau-positive cytoskeletal abnormalities in neurons and glial cells of aged baboons. The brains of four baboons (Papio hamadryas, 20-30 yr of age) were examined using the Gallyas silver technique for neurofibrillary changes and phosphorylation-dependent anti-tau antibodies (AT8, AT100, AT270, PHF-1, TG-3). Conspicuous changes were noted in two animals, 26 and 30 yr of age. In both animals, a combination of neuronal and glial cytoskeletal pathology was seen preferentially affecting limbic brain areas, including the hippocampal formation. In the 30-yr-old animal, numerous tau-positive inclusions were seen in the granule cells of the fascia dentata. These cells even exhibited an accumulation of argyrophilic neurofibrillary tangles. The glial changes affected both astrocytes and oligodendrocytes. Tau-positive astrocytes were seen in perivascular, subpial, and subependymal locations. Tau-positive oligodendrocytes preferentially occurred in limbic fiber tracts including the entorhinal perforant path. Ultrastructurally, tau-positive straight filaments (10-14 nm) in both neurons and glial cells were revealed by anti-tau immunoelectron microscopy. This study thus indicates the potential usefulness of aged baboons for experimental investigation of neuronal and glial filamentous tau pathology. This nonhuman primate species may provide valuable information pertinent to the broad spectrum of human tauopathies.

Fate of developing astrocytes in the optic nerve of the myelin-deficient rat.

There is considerable debate on the development of a glial cell line in the rat optic nerve, which is characterized by the specific expression of the A2B5 and HNK-1 epitopes. This cell line has been assumed to give rise to oligodendrocytes and so-called type 2 astrocytes. However, it is doubtful that the latter cell type really exists in vivo. In the present study, we have addressed this question by investigating the development of astrocytes in the myelin-deficient (md) rat, which is characterized by dysmyelination and loss of oligodendrocytes. Defective oligodendrocytes were observed by the third postnatal day, well before the generation of type 2 astrocytes. Consequently, the number of type 2 astrocytes was reduced in cultures prepared from optic nerves of md rats vs. controls. This finding was not paralleled in vivo; i.e., no dying astrocytes were observed in md sections by conventional electron microscopy. However, immunoreactivity against the HNK-1 epitope was enhanced in md compared to control sections. Ultrastructurally, HNK-1 immunoreactivity was detected predominantly on the axonal surface at astroaxonal contact sites, which were found only at the nodes of Ranvier within controls but extended to the whole axonal surface in md animals. Only a minor portion of the immunoreactivity derived from glial cells, presumably from oligodendrocytes at the paranodal region in controls. Thus, the HNK-1 epitope is not a useful antigen for distinguishing astrocytes in the rat optic nerve. Accordingly, our results do not provide evidence for the existence of specialized type 2 astrocytes in vivo. In vitro, these cells are probably only oligodendrocytes that mimic some astroglial features if grown in serum-containing media.

CD15-containing glycoconjugates in the central nervous system.

CD15-containing glycoconjugates have a common trisaccharide residue, 3-fucosyl-N-acetyllactosamine, which can be recognized by a panel of monoclonal antibodies. Immunohistochemical studies revealed a widespread distribution of CD15 in several epithelial non-neural tissues as well as in the CNS. In the mature mammalian brain CD15-containing glycolipids and glycoproteins are constantly present in astrocytes, whereas oligodendrocytes and particular subpopulations of neurons are variably immunostained. CD15 immunoreactive astrocytes are spatially distributed in some brain regions, which points to specialized functions of astroglial subpopulations. The expression of CD15 follows a timely ordered pattern during the development of glial cells and neurons of certain brain areas, such as the human and rat cerebellum and the mouse visual system. During morphogenesis, CD15 may exert either growth-promoting or growth-repulsive activities to guide cell migration. In CNS lesions altered expression patterns of CD15 may occur. For example, in human gliomas the staining intensity for CD15 inversely correlates with the grade of malignancy. In degenerative brain diseases reactive astrocytes may reveal an increased labelling intensity on their cell surface as well as an abnormal cytosolic accumulation of the epitope. The functional significance of CD15 in the CNS is not exactly known yet. The carbohydrate could be involved in cellular adhesion and/or as receptor molecule in signal transduction pathways, as has recently been demonstrated for leukocyte-platelet or leukocyte-endothelial cell interactions.

Cocultures of meningeal and astrocytic cells--a model for the formation of the glial-limiting membrane.

The glial-limiting membrane at the border of the central nervous system (CNS) consists of glial endfeet covered by a basal lamina. The formation of the glia limitans seems to be controlled by adjacent meninges but only little is known about this interaction. In the present study astrocytes and meningeal cells were investigated in vitro to see if cocultures of these cells can serve as a suitable model for the differentiation of the glial-limiting membrane and can be used to define the conditions under which the glial-limiting membrane develops. The following observations were made in cocultures of meningeal and astrocytic cells of two-day-old rats: (i) epithelioid astrocytes were transformed into stellate cells; (ii) single colonies of proliferating epithelioid astrocytes were generated; (iii) the area around these colonies becomes devoid of meningeal cells, which seem to form a circular border around the astroglial islands; (iv) from the glial colonies long thin glial processes grow towards the surrounding meningeal cells, terminating at the site of contact; (v) in the contact zone between meningeal cells and astrocytes irregular shaped deposits of electron dense material resembling a basal lamina were seen. These observations indicate that indeed a structure resembling a glial-limiting membrane develops in cocultures of meningeal and astrocytic cells. Its formation depends on the balance of growth promoting effects of meningeal cells on astrocytes and growth inhibiting effects of astrocytes on meningeal cells. Both activities can be enriched from conditioned media of pure astrocytic or meningeal cell culture. The proposed model of meningo-astrocytic cocultures may be a helpful instrument for further investigations on the formation of the glia limitans.

Demonstration of parathyroid hormone-related protein in meninges and its receptor in astrocytes: evidence for a paracrine meningo-astrocytic loop.

In contrast to the nervous and glial tissue of the adult rat brain the meninges are immunoreactive for parathyroid hormone-related protein (PTHrP), a hormone that binds with high affinity to the recently cloned PTH/PTHrP receptor. Accordingly immunoreactivity is found in cultured meningeal cells but not in astrocytes. In contrast, astrocytes but not meningeal cells synthesize the mRNA for the PTHrP receptor shown by reverse transcription of total RNA preparations and subsequent polymerase chain reaction with primers specific for the PTHrP receptor. The expression of the PTH/PTHrP receptor was confirmed by the dose-dependent activation of the adenylate cyclase in astrocytes and the rapid development of cellular processes following on incubation with PTHrP. We conclude that PTHrP secreted by meninges forms a paracrine meningo-astrocytic loop and may cause astrocytic differentiation, possibly involved in the formation of the glial limiting membrane.

Transforming growth factor beta 1 and parathyroid hormone-related protein control the secretion of dipeptidyl peptidase II by rat astrocytes.

Meninges synthesize parathyroid hormone-related protein (PTHrP) and transforming growth factor (TGF beta 1). Both factors control the activity of the glial enzyme dipeptidyl peptidase II (DPP II): TGF beta 1 induces the secretion of enzyme activity by cultivated astrocytes in a dose dependent manner. The maximal effect is achieved with a concentration of 10 ng/ml and is dependent on the time of incubation. PTHrP itself has no effect on the release of DPP II activity but considerably reduces the effect of TGF beta 1. It is assumed that DPP II influences the course of cicatrization after penetrating injuries of the brain and thus, meninges control glial scarring by the release of TGF beta 1 and PTHrP.

The carbohydrate epitope 3-fucosyl-N-acetyllactosamine is region-specifically expressed in astrocytes of the rat brain. Light- and electron-microscopical observations.

The carbohydrate epitope 3-fucosyl-N-acetyllactosamine (CD15) is involved in cell-to-cell recognition processes in various tissues. In the CNS of the adult rat, immunoreactivity for CD15 reveals a region-specific distribution pattern by light microscopy. In the present study we investigated the ultrastructural localization of CD15 in the rat brain using preembedding immunocytochemical methods. In addition we studied CD15 expression in cultured astrocytes from optic nerves of 11-day-old rats. In optic nerve sections, immunostaining was found on the surface of astrocytes at various contact sites, i.e. astrocyte-astrocyte, astrocyte-oligodendrocyte, astrocyte-axon myelin, and astrocyte-blood vessel contacts. Oligodendrocyte-oligodendrocyte contacts, however, were always negative. In the telencephalic cortex, CD15 immunoreactivity was found in glial cell processes around synapses and in the cerebellar cortex in Bergmann glial cells. In astrocytes grown in serum-containing medium, CD15 was expressed on the surface of fibroblast-like glial fibrillary acidic protein-positive astrocytes, which were identified as type 1 astrocytes as well as on process-bearing A2B5-positive cells, representing type 2 astrocytes. The present data support the assumption that in the adult rodent brain, CD15 is exclusively expressed by astrocytes. The in vivo distribution of this carbohydrate molecule on distinct astroglial contact sites supports the notion that CD15 could act in cell-to-cell recognition processes.

Dipeptidyl peptidase II in astrocytes of the rat brain. Meningeal cells increase enzymic activity in cultivated astrocytes.

Astrocytes grown in media conditioned by meningeal cells (MCM) develop cellular processes and markedly increased protein per cell. One protein component affected is the dipeptidyl peptidase II (DPP II). The increase of DPP II activity is dose- and time-dependent and can also be elicited by the second messenger cAMP. More mature astrocytes express higher levels of DPP II than immature proliferating astrocytes. The rate of proliferation of astrocytes is markedly enhanced by enriched MCM. These observations lead to the assumption that DPP II has a function within the catabolic processes of cellular differentiation. To assess whether the in vitro results may reflect in vivo conditions, we investigated the postnatal development of DPP II in the rat brain. Differentiating astrocytes in vivo are especially found early postnatally and, indeed, during this period high specific activities are found in brain. Depending on the region investigated DPP II activities decrease within the first ten days to one fourth of their P2 level and finally reach at about similar levels in all brain regions. Exceptions are the hypothalamus, where the activity is generally 1.5- to 3-fold higher than elsewhere in brain, and pons and mesencephalon, where the perinatal activity peak is lacking. The bulk activity of DPP II in immature rat brains is attributed to differentiating astrocytes loosing it in later postnatal stages due to a neuronal influence.

Interleukin-1 beta does not induce reactive astrogliosis, neovascularization or scar formation in the immature rat brain.

The adult mammalian central nervous system (CNS) reacts to a penetrating injury with the formation of a glial scar consisting of a newly formed glia limitans accessoria, basement membrane and meningeal fibroblasts. By contrast, in fetal and perinatal mammals a similar injury evokes only a reduced reactive astrogliosis, and a typical astroglial scar begins to develop only when the lesion has been placed beyond a critical developmental period. In the present investigation we have tested the hypothesis that IL-1 beta plays a pivotal role in the process of cicatrization, by investigating whether immature animals develop a glial scar after IL-1 beta is injected into their CNS. Adult female rats were given injections of 2U recombinant IL-1 beta or PBS alone in the contralateral cortex in identical positions of the cerebral hemispheres. Postnatal day 2 (P2) rats received injections of either 1U IL-beta or PBS into the lateral aspect of the frontal cortex on each side. The animals were sacrificed 4 and 14 days post injection and the perilesional area was assessed for astrogliosis (expression of GFAP-immunoreactivity and the activity of glutamine synthetase), neovascularization (laminin-immunoreactivity on blood vessels at the lesion site), and the formation of a gliomeningeal scar (GFAP- and laminin-immunoreactivity at the lesion site). Using similar criteria for the evaluation, we found that in adult animals some of the processes associated with cicatrization are augmented. In the immature animals, however, the formation of the glio-meningeal scar is not altered by IL-1 beta, i.e. it remains absent. We conclude that IL-1 beta augments some responses of the cells involved in wound healing in the adult CNS, but does not alter key mechanisms operative in the reaction of the brain to a penetrating injury, as shown by its inability to alter the stage specific response of the immature brain.

Activity of sorbitol dehydrogenase during development of the rat brain.

The postnatal development of the sorbitol dehydrogenase (SDH) activity in rat brain was investigated in different ontogenetic stages by a photometric assay. The specific activity increases from birth up to 45 days of age and then decreases again. The developmental pattern is similar in all brain regions investigated but differs in the peak activity: Brain regions containing large proportions of white matter, e.g. pons, develop activities of SDH which are up to two times higher than those in regions consisting predominantly of gray matter, e.g. cerebral cortex. These results suggest that SDH may be localized predominantly in astrocytes typical for white matter (i.e. fibrous astrocytes) and/or in oligodendrocytes but is not necessarily restricted to a specialized type of astrocyte. From the results it is concluded that the activity of SDH is regulated metabolically.

Purification of two dipeptidyl aminopeptidases II from rat brain and their action on proline-containing neuropeptides.

From the soluble and membrane fractions of rat brain homogenate, two enzymes that liberate dipeptides of the type Xaa-Pro from chromogenic substrates were purified to homogeneity. The two isolated dipeptidyl peptidases had similar molecular and catalytic properties: For the native proteins, molecular weights of 110,000 were estimated; for the denatured proteins, the estimate was 52,500. Whereas the soluble peptidase yielded one band of pI 4.2 after analytical isoelectric focusing, two additional enzymatic active bands were detected between pI 4.2 and 4.3 for the membrane-associated form. As judged from identical patterns after neuraminidase treatment, both peptidases contained no sialic acid. A pH optimum of 5.5 was estimated for the hydrolysis of Gly-Pro- and Arg-Pro-nitroanilide. Substrates with alanine instead of proline in the penultimate position were hydrolyzed at comparable rates. Acidic amino acids in the ultimate N-terminal position of the substrates reduced the activities of the peptidases 100-fold as compared with corresponding substrates with unblocked neutral or, especially, basic termini. The action of the dipeptidyl peptidase on several peptides with N-terminal Xaa-Pro sequences was investigated. Tripeptides were rapidly hydrolyzed, but the activities considerably decreased with increasing chain length of the peptides. Although the tetrapeptide substance P 1-4 was still a good substrate, the activities detected for the sequential liberation of Xaa-Pro dipeptides from substance P itself or casomorphin were considerably lower. Longer peptides were not cleaved. The peptidases hydrolyzed Pro-Pro bonds, e.g., in bradykinin 1-3 or 1-5 fragments, but bradykinin itself was resistant. The enzymes were inhibited by serine protease inhibitors, like diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, and by high salt concentrations but not by the aminopeptidase inhibitors bacitracin and bestatin. Based on the molecular and catalytic properties, both enzymes can be classified as species of dipeptidyl peptidase II (EC 3.4.14.2) rather than IV (EC 3.4.14.5). However, some catalytic properties differentiate the brain enzyme from forms of dipeptidyl peptidase II of other sources.

Rat peritoneal mast cells release dipeptidyl peptidase II.

Purified rat peritoneal mast cells have a 10-20-fold higher dipeptidyl peptidase II (DPP II) activity as compared with that of macrophages from the same source. Upon stimulation with the secretagogue Compound 48/80, DPP II is released from peritoneal-lavage cells and from purified mast cells, but not from purified macrophages, in a dose-dependent manner. Maximally, about one-third of the DPP II present in peritoneal-lavage cells is released. Substance P and the antigen/IgE system probably produce a similar effect. Both histamine and Zn2+, two ingredients of mast-cell granules, strongly inhibit DPP II at concentrations reported to occur in the granules. A possible role of mast-cell DPP II in the remodelling of connective tissue is discussed.