Yongchang Qian

Astroglia as Metal Depots: Molecular Mechanisms for Metal Accumulation, Storage and Release

The brain is an organ that concentrates metals, and these metals are often localized to astroglia. An examination of metal physiology of brain cells, particularly astroglia, offers insights into the developmental neurotoxicity of certain metals, including lead (Pb), mercury (Hg), manganese (Mn), and copper (Cu). Xenobiotic metals probably accumulate in cells by exploiting the normal functions of proteins that transport and handle essential metals. In addition, essential metals may become toxic by accumulating at levels that exceed the normal metal buffering capacity of the cell. This review considers the uptake, accumulation, storage, and release of two xenobiotic metals, Pb and Hg, as well as two essential nutrient metals that are neurotoxic in high amounts, Mn and Cu. Evidence that each metal accumulates in astroglia is evaluated, together with the mechanisms the host cell may invoke to protect itself from cytoxicity.

Copper handling by astrocytes: insights into neurodegenerative diseases.

Copper (Cu) is an essential trace element in the brain that can be toxic at elevated levels. Cu accumulation is a suspected etiology in several neurodegenerative diseases, including Alzheimers disease, Parkinsons disease, and prion‐induced disorders. Astrocytes are a proposed depot in the brain for Cu and other metals, including lead (Pb). This article describes the physiological roles of Cu in the central nervous system and in selected neurodegenerative diseases, and reviews evidence that astrocytes accumulate Cu and protect neurons from Cu toxicity. Findings from murine genetic models of Menkes disease and from cell culture models concerning the molecular mechanisms by which astrocytes take up, store, and buffer Cu intracellularly are discussed, as well as potential mechanistic linkages between astrocyte functions in Cu handling and neurodegenerative diseases.

Lead-Induced Endoplasmic Reticulum (ER) Stress Responses in the Nervous System

Lead (Pb) poisoning continues to be a significant health risk because of its pervasiveness in the environment, its known neurotoxic effects in children, and potential endogenous exposure from Pb deposited in bone. New information about mechanisms by which Pb enters cells and its organelle targets within cells are briefly reviewed. Toxic effects of Pb on the endoplasmic reticulum (ER) are considered in detail, based on recent evidence that Pb induces the expression of the gene for 78-kD glucose-regulated protein (GRP78) and other ER stress genes. GRP78 is a molecular chaperone that binds transiently to proteins traversing through the ER and facilitates their folding, assembly, and transport. Models are presented for the induction of ER stress by Pb in astrocytes, the major cell type of the central nervous system, in which Pb accumulates. A key feature of the models is disruption of GRP78 function by direct Pb binding. Possible pathways by which Pb-bound GRP78 stimulates the unfolded protein response (UPR) in the ER are discussed, specifically transduction by IRE1/ATF6 and/or IRE1/JNK. The effect of Pb binding to GRP78 in the ER is expected to be a key component for understanding mechanisms of Pb-induced ER stress gene expression.

Induction of 78 kD glucose-regulated protein (GRP78) expression and redox-regulated transcription factor activity by lead and mercury in C6 rat glioma cells

Lead (Pb) and mercury (Hg) are widespread environmental contaminants that induce prominent neural toxicity. Although the brain is not the major Pb and Hg depot in the body, these metals preferentially accumulate in astroglia to exert toxic effects. In this study, we examined the effects of Pb acetate and HgCl2 on the expression of GRP78, a molecular chaperone in the endoplasmic reticulum (ER) that may provide cytoprotection in response to cellular stresses in the C6 rat glioma cell line. We also evaluated the DNA binding activities of several redox-regulated transcription factors in metal-treated cells. Our results showed that mRNA levels of GRP78 were up-regulated by Pb and Hg at 0.1 and 1 μM, but down-regulated at higher concentrations (10 μM). GRP78 protein levels increased in a concentration- and time-dependent manner in Pb and/or Hg-treated cells. Pb increased protein binding to the GST-Ya antioxidant/electrophile response element (ARE/EpRE) and to the NF-kB consensus binding sequence of the cytomegalovirus 2 (CMV2) promoter, but decreased protein binding to the Ha-ras ARE/EpRE or to the c-fos 12-O-tetradecanoyl-phorbol-13-acetate (TPA) response element (TRE). In contrast, Hg activated DNA binding by all redox-regulated transcription factors. These studies shed some light on the molecular mechanisms of Pb and Hg toxicity in C6 rat glioma cells and suggest that GRP78 and oxidative stress may participate in the neurotoxic response to these metals.

Functional analysis of copper homeostasis in cell culture models: a new perspective on internal copper transport.

The movement of copper ions across membrane barriers of vital organs and tissues is a priority topic in nutrition and one for which there continues to be little understanding of the mechanism. Reports of membrane-bound, copper-transporting adenosine triphosphatases (Cu-ATPases) selective for copper ions have brought new focus to the problem and prompted fresh ideas. Using a cell culture model approach, we attempted to learn whether transport into and out of cells depends on a Cu-ATPase. Measurement of transport kinetics in fibroblasts, brain glial cells, neuroblastoma cells, and placental cells showed differences in the rates of copper uptake and response to sulfhydryl reagents. BeWo cells, a human choriocarcinoma placental cell line, behaved as did Menkes fibroblasts by avidly absorbing copper but not releasing copper to the immediate environment. Further tests showed that BeWo cells did not express the transcript for the membrane-bound Cu-ATPase that has been identified with Menkes syndrome. Transcript induction, however, was achieved by growing BeWo cells on porous filters that allowed apical and basolateral surfaces to form. With transcript expression, the cells showed a capacity to release copper into the medium. BeWo cells also synthesized a form of ceruloplasmin whose structure differed from that of the plasma protein and hence may be a product of a different gene. BeWo cells may also express the gene for Wilson disease, thus linking Menkes and Wilson proteins to maternal delivery of copper. We constructed a model in which both ATPases work in concert in a vesicle-based transport mechanism. The vesicle model may help us understand the transport of copper across the placenta and all cells in general.

Toxicity assessment of complex mixtures remains a goal.

One of the initial steps in remediating contaminated environments is to assess the human and ecological health risk associated with exposure to contaminants in a specific medium. Presented here are the results of a five-year study investigating the toxicity of simple and complex mixtures. A series of model compounds and simple mixtures including polycyclic aromatic hydrocarbons (PAHs), pentachlorophenol (PCP), and halogenated aliphatic hydrocarbons (HAHs) were analyzed. Mixture toxicity was studied using microbial genotoxicity assays and cytotoxicity assays with renal and neural cells. The majority of binary mixtures described here induced additive responses. A limited number of samples were identified where binary mixtures induced inhibitory effects. For example, benzo(a)pyrene (BAP) alone induced 30% renal cell death, whereas an equimolar dose of chrysene and BAP only produced 1.6% cellular death. In none of the mixtures tested did the mixture toxicity results deviate from the predicted results by an order of magnitude. The results from testing binary mixtures in this study indicate that the results did not deviate significantly from additivity. Complex mixture results were more difficult to interpret. The toxicity of complex mixtures could not be accurately predicted based on chemical analysis. This could be due to chemical interactions or due to the presence of unidentified chemicals, such as alkyl PAHs or high molecular weight PAHs that are not included in the standard risk assessment procedure. Even though the results from these in vitro studies indicate that additive assumptions will generally be appropriate for binary mixtures similar to the ones tested here, the risk associated with complex mixtures remains a challenge to predict. Before the results of toxicity testing can be used to adjust risk assessment calculations, it is important to fully appreciate the chemical composition and to understand the mechanism of observed chemical interactions in animals chronically exposed to low doses of chemical mixtures. This research was supported by ATSDR Grant no. ATU684505 and NIEHS SBRP Grant no. P42 ES04917.

The involvement of copper transporter in lead-induced oxidative stress in astroglia.

Lead (Pb), depositing primarily in astroglia in the brain, is a well-known neurotoxicant and a risk factor for neurologic disorders. Pb has been reported to induce oxidative stress by probably the disturbance of copper (Cu) homeostasis in astroglia. Thus, we hypothesized that Pb-induced oxidative stress is initiated by interfering with Cu transporter in astroglia. In this study, we observed Pb-induced oxidative stress as indicated by reactive oxygen species (ROS) augmentation and GRP78 and GRP94 protein induction, and it was parallel to Cu accumulation intracellularly by Pb. To further address Cu transporter as a potential Pb target, a heavy metal-binding (HMB) domain of Cu-transporting ATPase (Atp7a) was overexpressed and purified. Evidence showed that one molecule of HMB chelated 11 Pb ions or seven Cu ions and that Pb competed with Cu for binding to HMB. These findings suggest that Pb-induced oxidative stress results from the impairment of Cu metabolism by Pb targeting of Atp7a.

A Menkes P-type ATPase involved in copper homeostasis in the central nervous system of the rat.

We previously reported that copper efflux from C6 rat glioma cells was blocked by a brief exposure to sulfhydryl reagents p-chloromercuribenzoate (PCMB) and iodoacetamide as well as dicyclohexylcarbodiimide, suggesting the possible involvement of a Cu-transporting ATPase in the efflux mechanism. In this report, we show that copper efflux from PC12 cells, a neuron-like cell line established from rat adrenal pheochromocytoma, is also inhibited by PCMB exposure. Furthermore, we show that both C6 and PC12 cells express a homolog of the Menkes gene (MNK) as detected by RT-PCR with primers designed from a mouse cDNA and confirmed by sequence analysis of the amplified product. An expected 760-bp fragment representing the transduction and phosphorylation domains and a 925-bp fragment encoding the heavy metal-binding domain of Atp7a were amplified from a RNA extract of C6 and PC12 cells. Sequence data revealed that 690 bp of the 760-bp fragment from C6 cells were an identical match to a similar fragment from PC12 cells. Both fragments encoded a 229 amino-acid polypeptide that had a 98.7% sequence homology to mouse Atp7a. In addition, 880 bp from the 925-bp fragment of the two cell lines were identical and encoded a 293 amino-acid polypeptide with 94.5% sequence homology to mouse Atp7a. These data establish that a Menkes-type Cu-transporting ATPase is expressed in rat C6 and PC12 cells and strongly support the hypothesis that both neurons and glia are involved in maintaining Cu homeostasis in the central nervous system.

Involvement of the molecular chaperone Hspa5 in copper homeostasis in astrocytes

Copper (Cu) ion availability in tissues and cells must be closely regulated within safe limits by Cu transporters and chaperones. Astrocytes play key roles in metal homeostasis and distribution in the brain that are only partially understood. The purpose of this study was to define the role that the protein chaperone Hspa5, also known as Grp78, plays in Cu homeostasis in astrocytes. First passage cultures of primary astrocytes from neonatal rats and cultures of the C6 rat glioma cells were used as models. We found that the level of Cu accumulation in astrocyte cultures increased with Cu concentrations in the medium, and Cu treatment significantly reduced cellular levels of iron (Fe), manganese (Mn) and zinc (Zn). Cu accumulation specifically induced protein expression of Hspa5 but not metallothioneins (MTs) in astrocytes. In C6 cells, Hspa5 was identified as one component of a Cu-binding complex and shown to directly bind Cu. However, the level of Hspa5 expression was not proportional to Cu accumulation in astrocytes and C6 cells: astrocytes expressed low protein levels of Hspa5 compared to C6 cells but accumulated significantly more Cu than did C6 cells. Consistent with this finding, astrocytes expressed a lower level of the Cu-extruding protein Atp7a than did C6 cells, and depletion of Hspa5 by RNA interference resulted in significantly increased Cu accumulation and induction of MT1/2 expression. These data demonstrate that Hspa5 is involved in Cu homeostasis in astrocytes but not as a Cu storage protein.

Activation profiles of HSPA5 during the glomerular mesangial cell stress response to chemical injury

Abstract Environmental injury has been associated with endoplasmic reticulum (ER) stress, a response characterized by activation of the unfolded protein response, proteasomal degradation of proteins, and induction of HSPA5, also known as GRP78 or BiP. Although HSPA5 has been implicated in the stress response to environmental injury in several cell types, its role in the glomerular ER stress response is unknown. In this study, we evaluated HSPA5 activation profiles in rat glomerular mesangial cells (rGMCs) challenged with heavy metals (HgCl2 or Pb2+ acetate) or polycyclic aromatic hydrocarbons (PAHs, ie, benzo(a)pyrene [BaP]). Challenge of rGMCs with 1 or 10 μM HgCl2 or Pb2+ acetate increased HSPA5 mRNA and protein levels. The induction response was sensitive to transcriptional and translational inhibition by actinomycin D (AD) and cyclohexamide, respectively. HSPA5 mRNA was induced by 3 μM BaP in an AD-sensitive manner, but this response was unaffected by the presence of heavy metals. A promoter construct containing sequences that mediate thapsigargin (TH) inducibility of the HSPA5 promoter was refractory to both heavy metals and BaP. The HSPA5 induction response in rGMCs is conserved because it was reproduced with fidelity in immunolocalization experiments of HSPA5 protein in M15 and HEK293 cells in embryonic lines of murine and human origin, respectively. Collectively, these findings identify HSPA5 in the stress response of rGMCs and implicate regulatory mechanisms that are distinct from those involved in TH inducibility.