G. Perides

Neuroprotective effect of human osteogenic protein-1 in a rat model of cerebral hypoxia/ischemia

Possible neuroprotective actions of osteogenic protein-1 (OP-1) were evaluated in a rat model of cerebral hypoxia/ischemia. Intraperitoneal injection of 50 micrograms of OP-1 prior to bilateral carotid ligation and transient hypoxia in 12-day-old rats reduced cerebral infarct area from 44.8 +/- 3.3% in vehicle-injected controls to 29 +/- 4.9%. Treatment of 14-day-old rats with 20 micrograms of OP-1 1 h after hypoxia reduced mortality from 45% to 13%. OP-1 may represent a novel class of neuroprotective agents.

Co-localization of hyaluronic acid and chondroitin sulfate proteoglycan in rat cerebral cortex

The distribution of hyaluronate (HA) and chondroitin sulfate (CS) proteoglycan in the rat cerebral cortex was compared. For the localization of HA, the sections were incubated with human glial hyaluronate-binding protein (GHAP) and then reacted with monoclonal or polyclonal antibodies to GHAP. Polyclonal antibodies raised in rabbit were used for double-labeling experiments with monoclonal antibodies raised in mice and reacting with CS proteoglycans. Little reactivity was observed in rat cerebral cortex with polyclonal GHAP antibodies if the sections were not incubated with GHAP. Monoclonal antibodies to GHAP did not react with murine tissues. CS proteoglycans were localized in chondroitinase-digested sections with monoclonal antibodies reacting with the 4-sulfated oligosaccharide stubs formed by the digestion with chondroitinase ABC of CS side chains. In the rat cerebral cortex, the distribution of CS proteoglycans was similar to that reported by Bertolotto, A., Rocca, G. and Schiffer, D., J. Neurol. Sci., 100 (1990) 113-123, and his collaborators using the same antibodies. Many neurons mainly located in the upper and deep cortical layers were surrounded by CS immunoreactive material. Several (but not all) CS-positive neurons also stained for HA with an identical distribution except that in most instances the staining was confined to the periphery of the perikaryon and did not extend to the dendritic tree. The finding suggests that cerebral cortex CS proteoglycan is capable of interacting with HA.

β‐Amyloid peptide induces formation of actin stress fibers through p38 mitogen‐activated protein kinase

Based on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial β‐amyloid precursor protein APP‐V642I (IAPP) or mutant presenilin‐1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild‐type amyloid precursor protein or presenilin‐1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with β‐amyloid (Aβ) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a γ‐secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Aβ peptide induces actin stress fibers. Because activation of the p38 mitogen‐activated protein kinase (p38MAPK)–mitogen‐associated protein kinase‐associated protein kinase (MAPKAPK)‐2–heat‐shock protein 27 signaling pathway mediates actin polymerization, we explored whether Aβ peptide activates p38MAPK and MAPKAPK‐2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK‐2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Aβ peptide, IAPP or mPS1. Moreover, treatment with a γ‐secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK‐2. In summary, our data suggest that overproduction of Aβ peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.

Colocalization of tenascin with versican, a hyaluronate-binding chondroitin sulfate proteoglycan

Rabbit antisera against tenascin, a large extracellular matrix protein, in conjunction with monoclonal antibodies of mouse origin against versican, a large hyaluronate-binding proteoglycan, were used to make a comparative study of the distribution of the two antigens in the same cryostat sections by double immunofluorescence. In the central nervous system, tenascin was invariably associated with versican, but the reverse was not true, in that versican was also found where tenascin was not detectable, particularly in gray matter. There were major species differences in the distribution of tenascin in the central nervous system. In the cow, tenascin was found in cerebral and spinal cord white matter and in the granule cell layer of the cerebellum. In the human brain, tenascin was found in cerebral white matter but not in the cerebellum. In the rat, tenascin was mainly confined to brain periventricular layer and spinal cord white matter. During the development of the cerebellum of the rat, the tenascin immunoreactivity decreased, and a lower molecular weight band appeared (J1-160/180/restrictin?) and persisted throughout adulthood. Tenascin expression was a relatively late event in the development of the rat central nervous system, immunoreactivity being first observed after birth. In the rat embryo, tenascin was found to co-localize with versican in precartilaginous mesenchyme and in connective tissue underlying epithelia. The colocalization of versican with tenascin suggests that versican may be the tenascin (cytotactin)-associated proteoglycan reported in the literature.

The extracellular matrix of rat spinal cord: A comparative study on the localization of hyaluronic acid, glial hyaluronate-binding protein, and chondroitin sulfate proteoglycan

The localization of hyaluronic acid (HA), glial hyaluronate-binding protein (GHAP), and chondroitin sulfate (CS) proteoglycan was compared in cryostat sections of rat spinal cord. HA, GHAP, and CS proteoglycan were similarly distributed in white matter where they surrounded myelinated axons. In gray matter, large motoneurons were surrounded by a rim of reaction product in sections stained for HA and CS proteoglycan. GHAP immunoreactivity as well as HA had disappeared in hyaluronidase-digested sections, while CS proteoglycan immunoreactivity was not abolished under these conditions.

The extracellular matrix of cerebral gray matter : Golgi's pericellular net and Nissl's Nervösen Grau revisited

Glial hyaluronate‐binding protein (GHAP) and a large aggregating chondroitin sulfate proteoglycan (Ag‐Pg) similar to a fibroblast proteoglycan (versican) were localized in bovine, dog and cat central nervous system (CNS) gray matter by indirect immunofluorescence. The distribution of the two hyaluronate‐binding proteins was identical with that of hyaluronate, an extracellular glycosaminoglycan. All substances formed a finely reticulated mesh in the neuropil with a condensation of the stain around large neurons. It is concluded that in gray matter, as in white matter, the extracellular matrix (ECM) contains hyaluronate‐protein aggregates. We suggest that the hyaluronate‐protein aggregates correspond to the pericellular network first described by Golgi.

The astrocyte--extracellular matrix complex in CNS myelinated tracts: a comparative study on the distribution of hyaluronate in rat, goldfish and lamprey.

SummaryThe localization of hyaluronate was studied in the CNS of rat, goldfish and lamprey. Cryostat sections were incubated with glial hyaluronate-binding protein of human origin and stained by indirect immunofluorescence with glial hyaluronate binding protein antibodies not reaching with rat and fish. As previously reported for glial hyaluronate-binding protein and glial fibrillary acidic protein, hyaluronate and glial fibrillary acidic protein had a similar distribution in rat spinal cord and optic nerve, both substances forming ring-like structures around individual myelinated axons. A similar periaxonal distribution was observed in goldfish spinal cord and medulla, except that the rings were much wider, to accommodate the large goldfish axons. The glial fibrillary acidic protein-positive neuroglial tissue forming distinctive structures in goldfish vagal lobes also stained for hyaluronate. In both rat and goldfish spinal cord, motoneurons were surrounded by a hyaluronate coat. Goldfish optic nerve and lamprey spinal cord were hyaluronate-negative and, as previously reported, they stained for keratin but not for glial fibrillary acidic protein. The findings suggest that hyaluronate in CNS fibre tracts in a product of glial fibrillary acidic protein-positive neuroglia. They also suggest that the appearance of glial fibrillary acidic protein-positive neuroglia and the formation of a hyaluronate-bound extracellular matrix are related phenomena in phylogeny.

Axonal regeneration in old multiple sclerosis plaques

SummaryCryostat sections of two old plaques removed at autopsy from the spinal cord of a 62-year-old man with multiple sclerosis of 24-year duration were studied by indirect immunofluorescence with antibodies to neurofilament proteins, glial fibrillary acidic protein (GFAP), glial hyaluronate-binding protein (GHAP), vimentin and laminin. The neurofilament monoclonal antibodies used in this study reacted with phosphorylated epitopes of the two large polypeptides of the neurofilament triplet (NF 150K, NF 200K). As previously reported [Dahl D, Labkovsky B, Bignami A (1989) Brain Res Bull 22:225–232], the neurofilament antibodies either stained axons in the distal stump of transected sciatic nerve in the early stages of regeneration or late in the process, i.e., after regenerating axons had reached the distal stump of the transected sciatic nerve. Both multiple sclerosis plaques were positive for GFAP and vimentin, but negative for GHAP, while astrocytes in myelinated spinal cord white matter stained with both GFAP and GHAP antibodies. Laminin immunoreactivity in the plaques and normal spinal cord was confined to blood vessels. One plaque was almost devoid of axons as evidenced by indirect immunofluorescence with neurofilament antibodies. Another plaque was packed with bundles of thin axons running an irregular course in the densely gliosed tissue. Axons in the plaque only stained with neurofilament antibodies reacting with sciatic nerve in the early stages of regeneration while axons in the surrounding myelinated white matter were decorated by all neurofilament antibodies, regardless of the time of appearance of immunoreactivity in crushed sciatic nerve. It is concluded that reactive astrocytes forming glial scars do not constitute a non-permissible substrate for axonal growth.

Interaction of a brain extracellular matrix protein with hyaluronic acid

A glial hyaluronate-binding protein (GHAP) was isolated from bovine spinal cord and partially characterized. Bovine GHAP consisted of three immunologically related polypeptides with molecular masses of 76, 64, and 54 kDa and isoelectric points of 4.1, 4.2, and 4.4, respectively. Peptide mapping and partial amino acid sequencing showed that all three polypeptides derive from the same protein. The protein was localized immunohistochemically with rabbit antisera in the white matter surrounding the myelinated axons. Sugar analyses indicated that the three polypeptides are glycosylated and the sugar residues account for at least 30% of their weight. After enzymatic deglycosylation, the apparent molecular mass of the bovine GHAP was reduced to 43 kDa. The biochemical properties of bovine GHAP were compared to those of human GHAP. Initial peptide mapping indicated similarities between bovine and human GHAP. Partial amino acid sequencing of bovine GHAP showed a striking identity (up to 90%) with human GHAP and with the hyaluronate binding domain of the large human fibroblast proteoglycan, versican. Bovine and human GHAP were demonstrated to bind specifically to hyaluronic acid (HA) with one protein molecule binding to an average 17 disaccharide repeating units. The binding of bovine and human GHAP was inhibited by oligosaccharides of HA and specifically by the octamer. Salt concentrations of up to 1 M NaCl had very little effect on the binding of the GHAP to HA. The GHAP-HA interaction was pH dependent. Dissociation only took place at low pH (less than 3.5). Analysis of several polypeptides derived from GHAP by limited proteolysis allowed us to conclude that one of the tandem repeated sequences is sufficient for HA binding and that the aminoterminal domain (which contains an immunoglobulin-like fold) is not involved in the GHAP-HA-binding event.

Brain extracellular matrix and nerve regeneration.

Over the past five years, we have made some progress in our studies on the composition of brain extracellular matrix. As in previous work on GFA protein, a major component of glial scars, the motivation for these studies was to find out why axons do not regenerate in mammalian CNS. In fact, we started doing research on brain extracellular matrix because the experimental evidence suggested that the glial scar per se, could not explain the riddle of CNS regeneration (Bignami et al., 1986).