Transport of prion protein across the blood-brain barrier

The cellular form of the prion protein (PrP^c) is necessary for the development of prion diseases and is a highly conserved protein that may play a role in neuroprotection. PrP^c is found in both blood and cerebrospinal fluid and is likely produced by both peripheral tissues and the central nervous system (CNS). Exchange of PrP^c between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications, but it is unknown whether PrP^c can cross the blood-brain barrier (BBB). Here, we found that radioactively labeled PrP^c crossed the BBB in both the brain-to-blood and blood-to-brain directions. PrP^c was enzymatically stable in blood and in brain, was cleared by liver and kidney, and was sequestered by spleen and the cervical lymph nodes. Circulating PrP^c entered all regions of the CNS, but uptake by the lumbar and cervical spinal cord, hypothalamus, thalamus, and striatum was particularly high. These results show that PrP^c has bidirectional, saturable transport across the BBB and selectively targets some CNS regions. Such transport may play a role in PrP^c function and prion replication.     

Hyperammonemia causes altered protein phosphorylation in astrocytes

Treatment of primary astrocyte cultures with ammonium chloride for one day prior to phosphoprotein labeling resulted in a reduction in phosphate incorporation in a 66-kDa protein. Increasing ammonium chloride concentrations (2, 5, and 10 mM) led to greater reductions in phosphate incorporation in this band. The specificity of the effect was indicated by the lack of change in phosphate incorporation in 7 other protein bands. These results indicate that protein phosphorylation can be affected by pathophysiological concentrations of ammonia and suggest that altered protein phosphorylation may be related to the pathogenesis of disorders such as hepatic encephalopathy and Reye's syndrome where ammonia has been implicated as an important etiological factor.     

Brain biopsy in Creutzfeldt-Jakob disease: evolution of pathological changes by prion protein immunohistochemistry

The formation of protease-resistant prion protein (PrPsc) is considered to be an early event in the pathogenesis of Creutzfeldt-Jakob disease (CJD) and hence its demonstration in brain biopsies by immunohistochemistry is considered diagnostic. We analysed eight brain biopsies from the frontal cortex collected from different parts of India from cases diagnosed as CJD on clinical and pathological grounds for the expression of prion protein (PrP). The duration of illness in these cases varied from 2 months to 1 year. Immunohistochemistry was carried out on paraffin sections using two different clones (KG9 and 3F4) of monoclonal antibodies to PrP. Although all eight cases showed classical features of spongiform encephalopathy of varying severity, only five of the eight cases revealed PrP(sc) in the brain tissue. The immunolabelling was focal and all areas with spongiform change were not labelled. A temporal evolution in the staining pattern was evident - particulate diffuse labelling (synaptic type) in early stages (2 months), perivacuolar deposits in intermediate stages (5-6 months), and dense plaques in late stages (12 months).     

Norepinephrine-mediated protein phosphorylation in astrocytes

Studies were conducted to determine if norepinephrine activates both protein kinase C and the cyclic AMP-dependent protein kinase in cultured rat astrocytes using phosphoproteins as markers. Norepinephrine was found to decrease 32P incorporation into an acidic 80,000 M(R) protein. A similar response was observed with isoproterenol and cyclic AMP analogs. In contrast, phorbol myristate acetate (PMA) increased 32P incorporation into this protein. Further studies looked at phosphorylation sites on glial fibrillary acidic protein and vimentin using two-dimensional tryptic phosphopeptide maps. The pattern of phosphorylation of these two proteins by norepinephrine resembles that of 8-bromo cyclic AMP and isoproterenol, and not that of PMA. Additionally, the effect of norepinephrine on the phosphorylation of GFAP and vimentin was blocked by alprenolol. One difference noted between norepinephrine and isoproterenol was the phosphorylation of an 18,000 M(R) protein. Norepinephrine increased, and isoproterenol decreased, 32P incorporation into this protein; however, the mechanism which mediates the norepinephrine effect remains to be determined. Overall, these studies indicate that the most prominent phosphorylation events mediated by norepinephrine are the consequence of the activation of cyclic AMP-dependent protein kinase.     

星形胶质细胞在三方突触中对突触信息的加工处理和调控作用

传统观念认为脑组织功能专属于神经元活动，最新研究表明除了经典的突触前、后神经元之间存在"双向"信息流之外，星形胶质细胞也参与突触的神经元间信息交换、对突触活性做出反应、调节突触传递等过程。本文将对星形角质细胞在突触生理学中的作用，就其整合和加工处理突触信息并通过释放胶质递质最终调节突触传递和可塑性综述如下。     

Deposition of disease-associated prion protein involves the peripheral nervous system in experimental scrapie

There is some evidence that the peripheral nervous system (PNS) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The TSE-specific abnormal prion protein (PrP^sc) is considered as surrogate marker for infectivity. We traced the deposition of PrP^sc by immunocytochemistry in sheep and hamsters inoculated intraperitoneally with scrapie. The trigeminal, dorsal root, celiac, thoracic, and nodose ganglia contained ganglion cells and fewer satellite cells with prominent granular PrP^sc deposition. As a novel deposition pattern, punctate deposits in adaxonal location were seen along nerve fibers of peripheral nerve adjacent to ganglia. Such prominent involvement of the PNS in two different experimental scrapie models emphasizes the need to consider the PNS in natural scrapie and other TSEs including bovine spongiform encephalopathy as potential source of infectivity. Received: 5 May 1999 / Revised, accepted: 5 July 1999     

Calcium-activated, phospholipid-dependent protein kinase and protein substrates in primary cultures of astrocytes

Phosphoinositide-linked transmembrane signaling in the brain involves calcium-activated, phospholipid-dependent protein kinase (protein kinase C), but little is known about the glial contribution to this system. We observed that phosphorylation of several proteins in a cytosal fraction of rat astrocytes in primary culture was increased by the addition of calcium and phosphatidylserine. These agents also stimulated phosphate incorporation into lysine-rich histone, a substrate for protein kinase C. Addition of diacylglycerol, an activator of protein kinase C, further increased histone phosphorylation, whereas polymyxin B, an inhibitor of protein kinase C, blocked the stimulatory effect of calcium and phosphatidylserine. Based on enzyme units per mg protein, the activity of protein kinase C in astrocytes appears similar to that in whole brain cytosol. These results indicate that astrocytes display protein kinase C activity and suggest that the glial enzyme may be an important component of the receptor-linked phosphoinositide response system in the brain.     

Prion disease with a 144 base pair insertion: unusual cerebellar prion protein immunoreactivity

Sporadic, acquired, and genetic human prion diseases are characterized neuropathologically by distinct deposition patterns of the abnormal, disease-associated form of the prion protein (PrP^sc). In addition to mutations in the prion protein gene (PRNP), PrP^sc immunostaining patterns correlate with molecular phenotypes of prion diseases defined by the PRNP polymorphism at codon 129 and with protease-resistant PrP classified by Western blotting. Some point or insertional PRNP mutations share similar clinical and neuropathological phenotypes, whereas others show great variability even within the same family. Here we report a patient who presented clinically as sporadic Creutzfeldt-Jakob disease (CJD). Histologically moderate spongiform change was seen in cerebral and cerebellar cortical areas. Neuronal loss was restricted mainly to the occipital cortex and the basal ganglia. Surprisingly, numerous eosinophilic globular structures were noted in the molecular layer and the parahippocampal gyrus. These globules showed intense PrP immunopositivity using anti-PrP antibodies against different epitopes. They were stained with PAS but lacked congophilia and birefringence in polarized light. Ultrastructurally, globules were composed of 21-nm-thick intermingled filaments without dense core. Genetic analysis revealed a PRNP 144 base pair insertion. Our case reinforces the importance of molecular genetic diagnosis, especially in those patients who lack a family history of prion disease and show unusual neuropathological changes. It also widens the phenotypic spectrum of prion diseases. The phenotypic variability within the same mutation suggests further, yet uncharacterized, genetic or epigenetic influence on phenotype in these diseases.     

Copper binding is the governing determinant of prion protein turnover

The cellular isoform of the prion protein (PrP(c)) is located at the cell membrane, anchored externally by a glycosylphosphatidylinositol (GPI) anchor. It is a copper (Cu) binding glycoprotein with a rapid basal turnover. Previous studies have shown that exposure of cells to Cu causes internalisation of PrP(c) in vitro. In this study, we show that physiological levels of Cu promote internalisation of PrP(c). Interaction between PrP(c) and Cu was found to be the overriding factor in stimulating the internalisation response with other metals showing no effect. Deletion mutation studies have shown that two domains are essential for copper-induced internalisation to occur. These two domains are the octameric repeat region, encompassing amino acids 51-89, and the palindromic region, amino acids 112-119 with the sequence AGAAAAGA. The decrease in detectable levels of PrP(c) at the cell surface following Cu treatment was found to be the result of rapid internalisation rather than loss into the surrounding environment. These results have implications for both normal metabolism of PrP(c) and the possible mechanism of conversion of PrP(c) to PrP(sc).     

Astrocytosis and amyloid deposition in scrapie-infected hamsters

In scrapie infection, prion protein (PrP^Sc) is localized in areas where there is neurodegeneration and astrocytosis. It is thought that PrP^Sc is toxic to neurons and trophic for astrocytes. In our study, paraffin sections from scrapie infected (263K and 139H) and control hamsters were examined with histological and immunocytochemical staining. We found that PrP^Sc was present in the ependymal cells of both 263K- and 139H-infected hamsters. In 139H-infected hamsters, PrP^Sc was found in the cytoplasm of neurons in cerebral cortex and in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. In contrast, neuronal cytoplasm and nuclei, were positive for PrP^Sc in most areas such as cortex, hippocampus, and thalamus in 263K-infected hamsters. Many aggregations of PrP^Sc could be seen in the cortex, hippocampus, substantia nigra and around the Pia mater, corpus callosum, fimbria, ventricles, and blood vessels in sections from 139H- and/or 263K-positive animals. Furthermore, PrP^Sc was also co-localized with glial fibrillary acidic protein (GFAP) in many reactive astrocytes (approximately 90%) in certain areas such as the hippocampus in 263K-infected hamsters, but not 139H-infected hamsters. The patterns of astrocytosis and PrP^Sc formation were different between 139H- and 263K-infected hamsters, which may be used for a diagnosis purpose. Our results suggest a hypothesis that multiple cell-types are capable of PrP^Sc production. Our results also confirm that reactive astrocytes can produce and/or accumulate PrP^Sc during some scrapie strain infections. The findings suggest a `snowball effect', that is: astrocytosis might play an important role in amyloidosis, while amyloidosis may induce further astrocytosis at least in 263K-infected hamsters.     

Advances in the detection of prion protein in peripheral tissues of variant Creutzfeldt-Jakob disease patients using paraffin-embedded tissue blotting

The accumulation of PrP(Sc), an abnormal and disease-associated form of the normal prion protein (PrP(c)), within the central nervous system (CNS) is a key pathological feature of Creutzfeldt-Jakob disease (CJD). Following limited proteolytic digestion of PrP(Sc), the detection of PrP(res) within lymphoid tissues is a unique characteristic of variant CJD in comparison with other human prion diseases, raising fears of an increased risk of iatrogenic spread. Because levels of PrP(res) in lymphoid tissues are lower than those found in CNS tissue, there is concern that other peripheral tissues may harbour infectivity at levels that current detection systems cannot demonstrate PrP(res). We have modified the paraffin-embedded tissue blot (PET blot), a technique combining immunohistochemistry (IHC), histoblot and Western blotting, for the detection of PrP(res) in paraffin sections in peripheral tissues in variant CJD. Five cases of variant CJD were examined, using a panel of anti-PrP antibodies. In each of these five cases, spleen, tonsil, lymph nodes and dorsal root ganglia showed an increase in the sensitivity and specificity of labelling using the PET blot when compared with optimized PrP(res) IHC methods. Control cases showed no evidence of PrP accumulation in either peripheral or CNS tissues. Autopsy and biopsy brain material from sporadic CJD cases also showed an increased sensitivity of PrP(res) detection with the PET blot, confirming its value as an important diagnostic and research tool in human prion diseases.     

A novel method of generating neuronal cell lines from gene-knockout mice to study prion protein membrane orientation

The technology of gene knockout and transgenic mice has allowed the study of the role of genes and their proteins in animal physiology and metabolism. However, these techniques have often been found to be limited in that some genetic manipulations of mice led either to a fatal phenotype or to compensations that mask the loss of function of the target protein. The experimentation on neurons from transgenic mice is particularly critical in the study of key proteins that may be involved in neurodegeneration. The cell fusion technique has been implemented as a novel way to generate cell lines from prion protein knockout mice. Fusion between neonatal mouse neurons and a neuroblastoma cell line have led to a Prnp degrees / degrees cell line that facilitates the study of the knockout phenotype. These cells are readily transfectable and allowed us to study the expression of prion protein mutants on a PrP-knockout background. Using this cell line we have examined the effect of PrP mutations reported to alter PrPc to a transmembrane form. Our results suggest that these mutations do not create transmembrane forms of the protein, but block normal transport of PrP to the cell membrane.     

Mechanotransducing ion channels in astrocytes

Ion channels present on the soma of neonatal rat astrocytes in primary cell culture were studied using the single channel recording technique. Ion channels were activated by changing the pressure in the back of the pipette. The morphological structure of the patch membrane was examined while recording channel activity. One class of channel was activated by increasing the pipette pressure (curvature-sensitive or CS channels). CS channels were observed in 150 mM KCl, 150 mM NaCl, or 150 mM sodium gluconate. At constant pressure the closed times decreased with depolarization. CS channels had a conductance of 50 pS in 150 mM NaCl, and displayed an inwardly rectifying current-voltage relationship. CS channel activity was found only in cell-attached patches, and were active only when the patch membrane curved towards the soma. The other class of channel was found to be activated by both suction and pressure (stretch-activated or SA channels). Four SA conductance levels were found: 360, 230, 144, and 70 pS in 150 mM KCl. Each conductance displayed a linear current-voltage relationship. At negative membrane potentials SA channels were inhibited by Cs+, Ba2+ or Na+. The relationship between average mechanosensory current and pressure was biphasic for SA channels and monophasic for CS channels. Combinations of SA and CS channels could be observed in the same patch. We propose that CS channels are non-specific cation channels which sense membrane tension only when the patch membrane is in a specific, permissive curvature. SA channels appear to be K(+)-selective channels that sense membrane tension independent of the direction of curvature.     

Heparin inhibits the growth of astrocytes in vitro

Heparin at a concentration of 100 μg/ml, inhibits neonatal rat astrocyte proliferation by 45%, and [³H]thymidine incorporation into DNA by 55% when they are stimulated with 2% fetal bovine serum (FBS) in culture. Higher serum concentrations up to 10% FBS decreased this inhibition to 20% and 29% respectively. Inhibition of [³H]thymidine incorporation by heparin is dose-dependent with maximal inhibition at 100 μg/ml, but 39% inhibition is still seen at 1 μg/ml on stimulation by 5% FBS. Heparin or heparin-like molecules in the extracellular matrix of brain capillary walls may be important in the regulation of astrocyte growth in vivo.     

Growth factor pre-treatment differentially regulates phosphoinositide turnover downstream of lysophospholipid receptor and metabotropic glutamate receptors in cultured rat cerebrocortical astrocytes

Reactive gliosis is an aspect of neural plasticity and growth factor (GF) stimulation of astrocytes in vitro is widely regarded as a model system to study astrocyte plasticity. Astrocytes express receptors for several ligands including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), agonists for the G-protein-coupled lysophospholipid receptors (lpRs). Activation of lpRs by LPA or S1P leads to multiple pharmacological effects including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK), release of arachidonic acid, and induces mitogenesis. Treatment of astrocytes in vitro with a growth factor cocktail (containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF] and insulin) led to a marked attenuation of lpR-induced PI hydrolysis. In contrast, under identical conditions, GF treatment led to marked potentiation of PI hydrolysis downstream of activation of another abundantly expressed G-protein coupled receptor, mGluR5. Quantitative gene expression analysis of GF-treated or control astrocytes by TaqMan RT-PCR indicated that GF treatment did not change gene expression of lpa1 and s1p1, but increased gene expression of s1p5 which is expressed at very low levels in basal conditions. These results suggest that GF differentially affected PLC activation downstream of mGluR5 versus lpR activation and that the changes in mRNA levels of lpRs do not account for marked attenuation of agonist-induced phosphoinositide turnover.     

Prion protein on astrocytes or in extracellular fluid impedes neurodegeneration induced by truncated prion protein

Prion protein (PrP) is a host-encoded membrane-anchored glycoprotein which is required for susceptibility to prion disease. PrP may also be important for normal brain functions such as hippocampal spatial memory. Previously transgenic mice expressing amino terminally truncated mouse PrP (Δ32–134) spontaneously developed a fatal disease associated with degeneration of cerebellar granular neurons as well as vacuolar degeneration of deep cerebellar and brain stem white matter. This disease could be prevented by co-expression of wild-type (WT) mouse PrP on neurons or oligodendroglia. In the present experiments we studied Δ32–134 PrP transgenic mice with WT PrP expression restricted to astroglia, an abundant CNS cell-type important for neuronal viability. Expression of WT PrP in astroglia was sufficient to rescue 50% of mice from disease and prolonged survival by 200 days in the other 50%. We also found that transgenic mice expressing full-length soluble anchorless PrP had increased survival by 100 days. Together these two results indicated that rescue from neurodegeneration induced by Δ32–134 PrP might involve interactions between neurons expressing truncated PrP and nearby astrocytes expressing WT PrP or extracellular fluid containing soluble WT PrP.     

Codon 129 polymorphism and the E200K mutation do not affect the cellular prion protein isoform composition in the cerebrospinal fluid from patients with Creutzfeldt–Jakob disease

The cellular prion protein (PrP^c) is a multifunctional, highly conserved and ubiquitously expressed protein. It undergoes a number of modifications during its post‐translational processing, resulting in different PrP^c glycoforms and truncated PrP^c fragments. Limited data are available in humans on the expression and cleavage of PrP^c. In this study we investigated the PrP^c isoform composition in the cerebrospinal fluid from patients with different human prion diseases. The first group of patients was affected by sporadic Creutzfeldt–Jakob disease exhibiting different PrP codon 129 genotypes. The second group contained patients with a genetic form of Creutzfeldt–Jakob disease (E200K). The third group consisted of patients with fatal familial insomnia and the last group comprised cases with the Gerstmann–Sträussler–Scheinker syndrome. We examined whether the PrP codon 129 polymorphism in sporadic Creutzfeldt–Jakob disease as well as the type of prion disease in human patients has an impact on the glycosylation and processing of PrP^c. Immunoblotting analyses using different monoclonal PrP^c antibodies directed against various epitopes of PrP^c revealed, for all examined groups of patients, a consistent predominance of the glycosylated PrP^c isoforms as compared with the unglycosylated form. In addition, the antibody SAF70 recognized a variety of PrP^c fragments with sizes of 21, 18, 13 and 12 kDa. Our findings indicate that the polymorphisms at PrP codon 129, the E200K mutation at codon 200 or the examined types of human transmissible spongiform encephalopathies do not exert a measurable effect on the glycosylation and processing of PrP^c in human prion diseases.     

左乙拉西坦和传统抗癫痫药体外诱导大鼠星形胶质细胞P糖蛋白表达的研究

目的研究左乙拉西坦(LEV)和传统抗癫痫药(AEDs)丙戊酸钠(VPA)、卡马西平(CBZ)对大鼠皮质星形胶质细胞P-糖蛋白(P-gp)的表达的影响。方法不同浓度(1、10、50、100μg·m L-1)的VPA、CBZ及LEV持续作用于培养的正常新生鼠大脑皮质星型胶质细胞,分别在给药后10、20和30d,用免疫细胞化学法检测P-gp的表达率。结果对照组即无药物作用的正常星形胶质细胞P-gp表达率在各时点均小于5%;CBZ组100μg·m L-1在20d,30d较对照组表达增高(P0.05),20d与30d两组间比较,P0.05,其他浓度与对照组比较P0.05;VPA组100μg·m L-1在30d时较对照组表达增高(P0.05),较20d时表达增高(P0.05),其他浓度较各时点对照组比较,P0.05。LEV组在各浓度,不同时点与对照组比较差异无统计学意义(P0.05)。结论高浓度CBZ、VPA可诱导星形胶质细胞P-gp的表达,而LEV不能诱导星形胶质细胞P-gp表达。     

Immuno-electron microscopical localization of vimentin and glial fibrillary acidic protein in mouse astrocytes and their precursor cells in culture.

Immuno-electron microscopy was used to localize the distribution of vimentin and glial fibrillary acidic protein (GFAP) in mouse astrocytes and their precursor cells in primary cultures. In astroblasts and astrocytes, vimentin and GFAP form intermediate filaments (IF), which are heteropolymers, as previously observed in gliomas. Astrocytes and their precursor cells may have IF composed of GFAP-vimentin heteropolymer or vimentin alone, but IF composed of GFAP only were not seen. It seems that the formation of IF that are GFAP-vimentin heteropolymers is a feature of normal astroglia development and that the ratio of GFAP to vimentin in these IF reflects the degree of differentiation and functional state of the cell.