Ronald D. Poretz

Oligodendroglia in developmental neurotoxicity.

The developing nervous system has been long recognized as a primary target for a variety of toxicants. To date, most efforts to understand the impact of neurotoxic agents on the brain have focused primarily on neurons and to a lesser degree astroglia as cellular targets. The role of oligodendroglia, the myelin-forming cells in the central nervous system (CNS), in developmental neurotoxicity has been emphasized only in recent years. Oligodendrocytes originate from migratory, mitotic progenitors that mature progressively into postmitotic myelinating cells. During differentiation, oligodendroglial lineage cells pass through a series of distinct phenotypic stages that are characterized by different proliferative capacities and migratory abilities, as well as dramatic changes in morphology with sequential expression of unique developmental markers. In recent years, it has become appreciated that oligodendrocyte lineage cells have important functions other than those related to myelin formation and maintenance, including participation in neuronal survival and development, as well as neurotransmission and synaptic function. Substantial knowledge has accumulated on the control of oligodendroglial survival, migration, proliferation, and differentiation, as well as the cellular and molecular events involved in oligodendroglial development and myelin formation. Recently, studies have been initiated to address the role of oligodendrocyte lineage cells in neurotoxic processes. This article examines recent progress in oligodendroglial biology, focuses attention on the characteristic features of the oligodendrocyte developmental lineage as a model system for neurotoxicological studies, and explores the role of oligodendrocyte lineage cells in developmental neurotoxicity. The potential role of oligodendroglia in environmental lead neurotoxicity is presented to exemplify this thesis.

Protein kinase C activation is required for the lead-induced inhibition of proliferation and differentiation of cultured oligodendroglial progenitor cells

Lead (Pb) is a common neurotoxicant of major public health concern. Previous studies revealed that cultured oligodendrocyte progenitor cells (OPCs) are highly vulnerable to Pb toxicity. The present study examines the effect of Pb on the survival, proliferation and differentiation of OPCs in vitro. Dose-response studies showed that> or = l5-10 microM Pb is cytotoxic to OPCs within 24 h. However, 1 microM of Pb was found to inhibit the proliferation and differentiation of OPCs without affecting cell viability. Pb markedly decreased the proliferative capability of OPCs and inhibited cell-intrinsic lineage progression of OPCs at a late progenitor stage. The Pb-induced decrease of proliferation and differentiation was abolished by inhibition of protein kinase C (PKC) with bisindolylmaleimide I, while the effect of the PKC-activating agent phorbol-12,13-didecanoate was potentiated by Pb. Furthermore, Pb exposure of OPCs caused the translocation of PKC from the cytoplasm to membrane without an increase in total cellular PKC enzymic activity. These results indicate that Pb inhibits the proliferation and differentiation of oligodendrocyte lineage cells in vitro through a mechanism requiring PKC activation.

In-vitro photocytotoxicity of lysosomotropic immunoliposomes containing pheophorbide a with human bladder carcinoma cells☆

Pheophorbide a is a photocytotoxic agent. To develop a tissue-specific, intracellularly targeted photoactive system, pheophorbide a was incorporated into immunoliposomes coated with a monoclonal antibody (T-43) directed against the T-24 bladder tumor cell line. The efficacy of this system was studied in vitro using the human bladder tumor cell line MGH-U1. Uptake and localization were determined by the fluorescence of the immunoliposome markers within biochemically resolved subcellular components. The results demonstrate localization of the immunoliposome markers within the lysosomes of the tumor cells. Specific monoclonal antibody enhancement of the immunoliposomes uptake by MGH-U1 cells was demonstrated by the use of soluble T-43 monoclonal antibody as a competitive inhibitor. Pheophorbide-a-loaded immunoliposomes were shown to be photocytotoxic towards MGH-U1 cells at concentrations equivalent to photosensitizer at 500 ng ml-1. Treated cells, when protected from light, showed no cytotoxicity. These results demonstrate that uptake of pheophorbide-a-containing immunoliposomes by target cells and subsequent delivery to the lysosomes cause photoactivated killing of tumor cells. The utilization of immunoliposomes for intracellular lysosomal targeting of photoactive drugs to tumor cells constitutes a potentially valuable approach to photodynamic therapeutics.

Antimutagenicity, cytotoxicity and composition of chlorophyllin copper complex.

The preparation of chlorophyllin copper complex (CCC), shown to be a tumor promoter in an animal model (Nelson, R.L. (1992) Chlorophyllin, an antimutagen, acts as a tumor promoter in the rat-dimethylhydrazine colon carcinogenesis model. Anticancer Res., 12, 737-740), also inhibits the activities of direct- and indirect-acting mutagens in the Salmonella assay and exhibits cytostatic and cytocidal effects toward myeloma cells. Data from elemental analyses, spectrophotometry and reversed-phase high-performance liquid chromatography indicate that CCC preparations generally used in antimutagenic/anticarcinogenic experiments are variable, complex mixtures of structurally distinct porphyrins lacking copper in some instances. This variability of the composition may be a cause for the differences reported for the tumor promotion activity of CCC.

The (1 → 3)-linked α-l-fucosyl group of the N-glycans of the Wistaria floribunda lectins is recognized by a rabbit anti-serum☆

An increasing number of plant glycoproteins have been shown to possess a characteristic N-glycan component containing a beta-(1----2)-linked D-xylose unit on the core beta-D-mannose unit, and an alpha-(1----3)-linked L-fucose unit on the asparagine-linked 2-acetamido-2-deoxy-D-glucose unit. Wistaria floribunda seeds have two distinct lectins; the erythroagglutinin, WFA, and the lymphocyte mitogen, WFM. Earlier studies indicated that both lectins belong to such a class of glycoproteins. We now report the complete structural analysis of Pronase glycopeptides derived from WFA. On the basis of chemical treatment of the glycopeptides, carbohydrate composition and methylation analysis of fluorescein-labeled glycopeptides, and their susceptibility to specific exoglycosidases, the structure of the WFA glycan was found to be, alpha-D-Manp-(1----6)-[beta-D-Xylp-(1----2)]- [alpha-D-Manp(1----3)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc-[ alpha-L- Fucp-(1----3)]-beta-D-Glcp-NAc-(1----N). Quantitative studies on the interaction of the original fluorescein-labeled glycopeptide and its specific degradation products with a rabbit anti-glycan antibody, developed against WFM, showed that the (1----3)-linked alpha-L-fucose unit is essential for interaction. Loss of the terminal alpha-D-mannosyl groups resulted in decreased, though detectable binding.

Two-dimensional poly(acrylamide) electrophoresis of fluoresceinated glycopeptides. Resolution and structural characterization of ovalbumin glycans.

The microheterogeneous mixture of fluoresceinated glycopeptides (FGPs) obtained from the single site of glycosylation of chicken ovalbumin was resolved by a combination of discontinuous electrophoresis in a high-density poly(acrylamide) gel (PAGE) for sizing, in conjunction with borate-PAGE. Two FGPs of similar size but with different mobilities in borate-PAGE were purified and characterized by sequential exoglycosidase digestion and sizing on the discontinuous PAGE system, as well as by methylation analysis. The two FGPs of identical size are distinct and have structures beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->3)-[beta-D-Glc pNAc-(1-->4)]-[beta-D-Glc pNAc-(1-->2)-alpha-D- Man p-(1-->6)]-beta-D-Man-p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R and alpha-D-Man p-(1-->2)-alpha-D-Man p-(1-->3 or 6)-[alpha-D-Man p-(1-->3)-[alpha-D-Man p-(1-->6)]-alpha-D-Man p-(1-->6 or 3)]-beta-D-Man p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R (R = Asn-(amino acids)-fluorescein). The results demonstrate that two-dimensional PAGE is applicable to the separation and characterization of complex mixtures of FGPs. The procedure is rapid, sensitive, and convenient for glycopeptide mapping, and for the purification and structural characterization of glycans. Furthermore, the FGPs can be characterized with affinity matrices, such as lectins, and by methylation analysis.

The R496H mutation of arylsulfatase A does not cause metachromatic leukodystrophy

Deficiency of arylsulfatase A (ARSA) enzyme activity causes metachromatic leukodystrophy (MLD). A number of ARSA gene mutations responsible for MLD have been identified. Recently, the R496H mutation of ARSA was proposed to be a cause of MLD (Draghia et al., 1997). We have investigated the R496H mutation and found this mutation at a relatively high frequency in an African American population (f = 0.09, n = 61 subjects). The ARSA enzyme activity in subjects with and without the R496H mutation was determined and found to be normal. It is therefore concluded that the R496H mutation of ARSA does not negatively influence the activity of ARSA and is not a cause of MLD. Hum Mutat 12:238–239, 1998.

Intracellular Distribution Of Porphyrin-Based Photosensitizers In In Vitro Cultured Human Bladder Tumor Cells.

The Increasing use of porphyrin-based compounds for photodynamic therapy raises concerns about the intracellular distribution of these substances in target cells. We are utilizing chlorophyll derivatives as photosensitizers and exploring their intracellular distribution in in vitro cultivated EJ human bladder tumor cells. The cells are exposed to these compounds delivered within a liposome-based carrier system as well as in the free form. High resolution subcellular fractionation approaches are employed to examine the cellular localization of the photodynamic agents. Free pheophorbide a and hematoporphyrin derivative localize, almost exclusively within the plasma membrane of cells exposed to the agents for 90 min. In contrast the pheophorbide a loaded liposomes are readily taken into lysosomes, presumably by endocytosis, if presented to the cells at 4° and then incubated with the cells at 37°. However cells exposed to the liposomes continuously at 37° accumulate the pheophorbide a in the plasma membrane. These studies indicate that untargeted liposomes exhibiting a decreased fusegenic tendency than employed in these studies may allow for an effective delivery of photodynamic agent to lysosomes.

The separation of lymphocyte subpopulations with lectins.

Wistaria floribunda agglutinin (WFA), Sophora japonica agglutinin (SJA) and Maclura pomifera lectin (MPL) were employed as immunofluorescent and leucoagglutinating reagents to study murine lymphocytes. WFA, which labels 90% of thymocytes, binds to only 57% of the splenocyte population. The latter subset corresponds to surface immunoglobulin bearing cells. Differential agglutination of splenocytes with this lectin results in the isolation of a WFA negative population which exhibits T-lymphocyte surface markers. The agglutinable splenocytes bind only 2.5 times more WFA than non-agglutinable cells suggesting that the preferential agglutination of B-splenocytes is due to a combination of reduced cell surface negative charge and increased number of lectin binding sites on these cells as compared to T-lymphocytes. Forty percent of splenocytes are positive for SJA and differential agglutination of splenocytes yields a population of SJA non-agglutinable cells that are not labeled by this lectin. The two populations fractionated by SJA are unrelated to T- and B-lymphocyte subsets. Differential agglutination of thymocytes by SJA yields a non-agglutinable group representing 42% of total thymocytes. Although the nature of the two thymocyte subsets discriminated by SJA remains unknown, this lectin appears useful in identifying and separating unique thymocyte and splenocyte populations.

Lead causes human fibroblasts to mis-sort arylsulfatase A.

Lead exposure causes cognitive and behavioral deficits in some children. We have proposed that the effects of single nucleotide polymorphisms (SNP) of the human pseudodeficient arylsulfatase A (ARSA) gene that result in reduced levels of the enzyme, and lead concentrations that decrease ARSA activity, culminate in cellular enzymic activity that is below a critical threshold required for the normal nervous system function. Human fibroblasts grown in the presence of lead acetate exhibit a 65% decrease in ARSA protein, resulting in a significant decrease in the ability to catabolize sulfatide in cells from individuals with the SNP(s) of pseudodeficient ARSA, but not those from subjects with the normal gene (Poretz et al., Neurotoxicology 21 (2000) 379). The present study examines the potential of lead to affect the biosynthesis, trafficking and turnover of ARSA in human fibroblasts. Fibroblasts, grown in 20 microM lead, displayed a 44--58% increase in the rate of proliferation. Lead caused a decrease of approximately 33% in the accumulation of newly synthesized intracellular ARSA. This difference was not due to increased rates of intracellular degradation of ARSA or decreased levels of ARSA mRNA. Lead, however, caused the newly synthesized enzyme to be trafficked through the secretion pathway, resulting in decreased amounts of the enzyme in intracellular compartments. Though lead exposure results in increased cellular proliferation, it appears to cause decreased intracellular steady-state levels of ARSA by affecting the sorting cues and/or mechanisms directing the enzyme to lysosomes.