Lewy bodies (LBs) are the characteristic inclusions of Parkinson's disease brain but the mechanism responsible for their formation is obscure. Lewy bodies (LBs) are composed of a number of proteins of which alpha-synuclein (α-SYN) is a major constituent. In this study, we have investigated the distribution patterns of synphilin-1 and parkin proteins in control and sporadic PD brain tissue by immunohistochemistry (IH), immunoblotting, and immunoelectron microscopy (IEM). We demonstrate the presence of synphilin-1 and parkin in the central core of a majority of LBs using IH and IEM. Using IH, we show an overlapping distribution profile of the two proteins in central neurons. Additionally, we show sensitivity of both endogenous synphilin-1 and parkin to proteolytic dysfunction and their co-localization in aggresomes formed in response to the proteasome inhibitor MG-132. We confirm that synphilin-1 and parkin are components of majority of LBs in Parkinson's disease and that both proteins are susceptible to proteasomal degradation.

Two mutations in the DJ-1 gene on chromosome1p36 have been identified recently to cause early-onset, autosomal recessive Parkinson's disease. As no information is available regarding the distribution of DJ-1 protein in the human brain, in this study we used a monoclonal antibody for DJ-1 to map its distribution in frontal cortex and substantia nigra, regions invariably involved in Parkinson's disease. Western blotting of human frontal cortex showed DJ-1 to be an abundant protein in control, idiopathic Parkinson's disease, cases with clinical and pathological phenotypes of Parkinson's disease with R98Q polymorphism for DJ-1, and in progressive supranuclear palsy (PSP) brains. We also showed that DJ-1 immunoreactivity (IR) was particularly prominent in astrocytes and astrocytic processes in both control and Parkinson's disease frontal cortex, whereas neurons showed light or no DJ-1 IR. Only occasional Lewy bodies (LBs), the pathological hallmarks of Parkinson's disease, showed faint DJ-1 IR, localized to the outer halo. In preclinical studies we showed that DJ-1 is expressed in primary hippocampal and astrocyte cultures of mouse brain. By 2D gel analysis we also showed multiple pI isoforms for DJ-1 ranging between 5.5-6.6 in both control and Parkinson's disease brains, whilst exposure of M17 cells to the oxidizing agent paraquat was manifested as a shift in pI of endogenous DJ-1 towards more acidic isoforms. We conclude that DJ-1 is not an essential component of LBs and Lewy neurites, is expressed mainly by astrocytes in human brain tissue and is sensitive to oxidative stress conditions. These results are consistent with the hypothesis that neuronal-glial interactions are important in the pathophysiology of Parkinson's disease.

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (α-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that α-SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to α-SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.

A number of major properties of endothelial cells (EC) at the blood-brain barrier (BBB) have been shown to be astrocyte-dependent. Whether analogous properties at the blood-nerve barrier (BNB) are induced and maintained by Schwann cells has not been investigated. As a preliminary investigation we have undertaken a comparative study of six EC membrane markers at the BBB and BNB and perineurium. Employing immunoblotting and immunocytochemistry the relative distribution between rat brain cortex and sciatic nerve was determined for the glucose transporter (GLUT-1), the transferin receptor (OX-26), the endothelial barrier antigen (EBA) and the OX-47 antigen. Using enzyme cytochemistry the same comparison was made for γ-glutamyl transpeptidase (GGTP) and alkaline phosphatase. By immunocytochemistry GLUT-1 was uniformly strongly represented in brain EC, nerve EC and perineurium. OX-26 was strongly positive in brain EC but present only in trace quantities in nerve EC and perineurium. EBA similarly showed strong positivity in brain EC and trace amounts in nerve EC but was absent from perineurium. OX-47 was present moderately in brain EC and perineurium but absent from nerve EC. Quantitative immunoblotting of brain and sciatic nerve homogenates showed statistically significant differences in the level of expression of EBA and OX-26 between the two tissues. Enzyme cytochemistry showed that GGTP was strongly positive in brain EC but absent from nerve EC and perineurium. Alkaline phosphatase stained strongly in brain and nerve EC and was absent from perineurium. In summary the six membrane markers were heterogeneously represented in nerve compared with brain. This pattern of distribution in the nerve cannot simply be accounted for by the absence of astrocytes and their inductive influences. Any inductive influences of Schwann cells require investigation.

Pial microvessels have several important blood-brain barrier (BBB) characteristics in common with cerebral microvessels, despite lacking their astrocytic ensheathment. We have therefore determined whether they have the same distribution of two enzymes, γ-glutamyl transpeptidase (GGTP) and alkaline phosphatase, both of which are known to be astrocyte-dependent. GGTP was absent from all rat pial microvessels but strongly present in brain cortical capillaries. Alkaline phosphatase was heterogeneously expressed in pial microvessels, including capillaries, but strongly positive in brain cortical capillaries. Diffusible, inductive factors produced by astrocytes could account for these differences in enzyme distribution between the two vessel types. Furthermore, differences in expression between the two markers may reflect their differing sensitivities to the astrocytic factors. Caution is urged in the common usage of the pial microvessel as a model system in BBB studies.

The oligosaccharide chains of cell surface and extracellular matrix glycoconjugates are essential for the biological properties of these molecules. We have, therefore, investigated carbohydrate residues in the rat cornea using biotinylated lectin--gold probes. Fixed corneas were removed and embedded in Lowicryl HM20 or LR White. Ultrathin sections were incubated in one of the lectins: Triticum vulgare (WGA), Canavalia ensiformis (Con A), Griffonia simplicifolia (GS-1), Limax flavus (LFA) and Allomyrina dichotoma (Allo A), followed by streptavidin--gold, or the sections were incubated in cationic colloidal gold. Semi-quantification of gold labelling was determined for corneal endothelium, Descemet's membrane, stroma and epithelium from electron micrographs. WGA and Con A binding sites were expressed either moderately or strongly through out the cornea, suggesting a preponderance of alpha-mannose and N-acetylglucosamine residues. A particular concentration of these sugars was found in Descemet's membrane. In contrast, GS-1 (specific for alpha-galactose) and Allo A (specific for beta-galactose) labelled all regions weakly. Sialic acid residues, as defined by LFA labelling and the expression of neuraminidase-sensitive cationic colloidal gold binding sites, were sparsely distributed throughout the stroma, Descemet's membrane and endothelium. In contrast, sialoglycoconjugates were found in significant concentrations in the epithelium. Electron microscopy proved useful in providing new information on the cellular and subcellular localization of these lectin binding sites. © Chapman & Hall

Pial microvessels have commonly been used in studies of the blood-brain barrier because of their relative accessibility. To determine the validity of using the pial microvessel as a model system for the blood-brain barrier, we have extended the comparison of pial and cerebral microvessels at the molecular level by a partial characterization of the glycocalyx of pial endothelial cells, in view of the functional importance of anionic sites within the glycocalyx. Rat optic nerves were fixed by vascular perfusion. Anionic sites on the endothelium were labelled with cationic colloidal gold by means of post- and pre-embedding techniques. The effects of digestion of ultrathin sections on subsequent gold labelling was quantified following their treatment with a battery of enzymes. Biotinylated lectins, viz. wheat germ agglutinin and concanavalin A with streptavidin gold, were employed to identify specific saccharide residues. The results demonstrate that the luminal glycocalyx of pial microvessels is rich in sialic-acid-containing glycoproteins. Neuraminidase, which is specific for N-acetylneuraminic (sialic) acid, and papain (a protease with a wide specificity) significantly reduce cationic colloidal gold binding to the luminal endothelial cell plasma membrane. Wheat germ agglutinin (with an affinity for sialic acid) binds more to the luminal than abluminal plasma membrane, whereas concanavalin A, which binds mannose, binds more to the abluminal surface. Similar results have been obtained for cerebral cortical endothelial cells. With respect to these molecular characteristics, therefore, the pial and cortical microvessels appear to be the same. However, since the two vessel types differ in other respects, caution is urged regarding the use of pial microvessels to investigate the blood-brain barrier.

(1) Neurogenic inflammation has been implicated in the pathogenesis of the vascular headaches of migraine and cluster headaches. (2) Dural blood vessels are both pain-sensitive and show neurogenic plasma extravasation. (3) Endothelial cell (EC) surface anionic sites appear to be a determinant of vascular permeability. We therefore examined the anionic sites of dural EC to determine whether they are different from those of pial and parenchymal vessels. Luminal anionic sites of rat optic nerve EC were labelled with cationic colloidal gold (CCG) and cationic ferritin (CF) and examined by electron microscopy. Employing a battery of enzymes, the effects of digestion of ultrathin sections on subsequent labelling with CCG was quantified using image analysis software. In addition, a gold-labelled lectin, wheat-germ agglutinin (WGA), was employed to locate specific saccharide residues. Of the enzymes with a narrow specificity, only neuraminidase substantially reduced CCG binding. Of the proteolytic enzymes, papain was most effective in reducing labelling. These results show that the luminal EC anionic sites are chiefly composed of sialoglycoproteins. The labelling with biotinylated WGA-streptavidin gold was similar to that with CCG without enzyme digestion. This suggests that WGA is binding to N-acetylneuraminic (sialic) acid residues and not to the neutral N-acetylglucosamine (since CCG would not label uncharged molecules). These results do not differ significantly from those for pial and parenchymal EC. It is therefore likely that factors other than anionic site molecular composition account for the susceptibility of dural vessels to neurogenic plasma extravasation. The relevance of these observations in an experimental animal model to the human clinical condition remains to be determined.

A monoclonal antibody to an antigen (EBA) expressed by neural endothelial cells (EC) was used to investigate any difference in the distribution of EBA between the CNS and PNS. Pre-embedding ultrastructural cytochemistry of rat sciatic and optic nerves was undertaken using anti-EBA, detected with a silver-enhanced gold-conjugated secondary antibody. LM immunocytochemical localisation of EBA was also performed using an HRP-conjugated secondary antibody. EC of pial and parenchymal optic nerve vessels were strongly immunopositive for EBA. Vessels of the dura were negative. At the EM level EBA was observed on the EC luminal surface. In contrast, EC of sciatic nerve were either negative or only weakly immunopositive. The molecular characteristics and function of EBA are largely unknown. Therefore the functional significance of the present findings remains to be determined.

Incorporated in the luminal glycocalyx of vascular endothelia (EC) are negatively charged microdomains (anionic sites). These sites are considered functionally important (a) in their interaction with circulating blood constituents, and (b) as a determinant of vascular permeability. The molecular composition of these EC sites, described for a number of tissues, has demonstrated a heterogeneity dependent on their anatomical location. Luminal anionic sites have not been characterized for EC of optic nerve. Optic nerves were removed from Sprague-Dawley rats previously fixed by vascular perfusion. EC anionic sites were labelled with the probes cationic colloidal gold (CCG) and cationic ferritin (CF), using the pre- and post-embedding techniques, and examined by electron microscopy. The effects of enzyme digestion of ultrathin sections on subsequent CCG labelling were determined using a battery of enzymes in association with the post-embedding technique. CCG labelling was quantified following each enzyme treatment using image analysis software. The biotinylated lectin wheat germ agglutinin (WGA) with streptavidin gold was also used to localize specific monosaccharide residues. The luminal front of intraneural EC showed a uniform labelling with CCG and CF which was greater than on the abluminal surface. Extracellular matrix components and basal laminae were moderately labelled. Digestion of tissue sections with heparitinase and trypsin had no significant effect on subsequent CCG labelling. Proteinase K was less effective than papain but both produced a significant reduction. Neuraminidase almost completely eliminated labelling. CCG binding to the luminal plasma membrane of optic nerve EC can be significantly reduced with proteolytic and glycolytic enzymes. The results demonstrate that sialoglycoproteins principally constitute these luminal EC anionic sites. Biotinylated WGA-streptavidin gold, which detects both N-acetylneuraminic (sialic) acid and N-acetylglucosamine, gave a similar pattern of labelling to CCG alone on the luminal versus abluminal EC fronts. These findings suggest that WGA is binding predominantly to N-acetylneuraminic acid residues since CCG would not label the neutral (uncharged) N-acetylglucosamine.

BACKGROUND--A rat endothelial barrier antigen (EBA) recognised by a monoclonal antibody has been shown to be expressed strongly by endothelial cells of brain capillaries possessing a blood-brain barrier and only weakly expressed by fenestrated brain vessels. METHODS--In this study immunocytochemical methods for light and electron microscopy were used to study EBA distribution in the eye and orbital tissues of the rat. RESULTS--Blood-ocular barrier vessels in the optic nerve, retina, iris, and some vessels in th choroid and ciliary body were immunopositive for EBA. By pre-embedding immunocytochemistry for electron microscopy the antigen was observed on the luminal endothelial cell surface. CONCLUSION--Surprisingly, some non-barrier vessels in the ciliary body and choroid expressed EBA suggesting that it may play a broader role in endothelial properties than previously recognised. The functional significance of EBA remains to be elucidated.

The role of endothelial cell anionic surface charge in the function of the blood-nerve barrier (BNB) was investigated in 16 Sprague-Dawley male rats. The polycation protamine sulphate (PS) was administered parenterally in 8 animals to neutralize the endothelial anionic charge. The effect of such neutralization on the permeability of the BNB to intravenously injected horseradish peroxidase (HRP), and on anionic sites labelling with cationic colloidal gold (CCG) and cationic ferritin (CF) were studied qualitatively and quantitatively. Eight control animals were similarly studied after parenteral administration of saline or buffer instead of PS. PS-treated animals showed a pronounced opening of the BNB to HRP, and a significant reduction of endothelial cell surface labelling with cationic probes. The association of a reduction in anionic sites with the breakdown of the BNB supports the view of an endothelial surface charge filter acting as a contributing factor to the normal function of the BNB. The possible role of charge in transcellular and paracellular transport routes is discussed.

The distribution of anionic microdomains has been described in cerebral vessels and more recently in capillaries of peripheral nerve. Evidence is accumulating that these sites play a role in the barrier function of vascular endothelia in the PNS and CNS. The chemical nature of anionic sites has been at least partly determined for cerebral vessels but not in peripheral nerve. This study reports our preliminary investigations to determine the nature of endothelial anionic sites in sciatic nerve. The effects of digestion of ultra-thin sections of nerve with a battery of proteolytic and glycolytic enzymes (papain, trypsin, proteinase K, hyaluronidase, heparinase, heparitinase and neuraminidase) on the distribution of anionic sites was determined using the label, cationic colloidal gold. Papain, a proteolytic enzyme of broad specificity, succeeded in removing the majority of cationic colloidal gold-binding sites on the luminal surface of vascular endothelia. In contrast trypsin and proteinase K were less effective, reflecting their narrower specificity. Hyaluronidase, heparinase and heparitinase did not significantly affect cationic colloidal gold-labelling. However, a considerable reduction in cationic colloidal gold-binding occurred following neuraminidase digestion. These results suggest that, as in cerebral vessels, sialic acid-containing glycoproteins are largely responsible for the negatively charged domains on the luminal membrane of endothelial cells in peripheral nerve.