Lead exposure disturbs ATP7B-mediated copper export from brain barrier cells by inhibiting XIAP-regulated COMMD1 protein degradation.

The brain barrier is an important structure for metal ion homeostasis. According to studies, lead (Pb) exposure disrupts the transportation of copper (Cu) through the brain barrier, which may cause impairment of the nervous system; however, the specific mechanism is unknown. The previous studies suggested the X-linked inhibitor of apoptosis (XIAP) is a sensor for cellular Cu level which mediate the degradation of the MURR1 domain-containing 1 (COMMD1) protein. XIAP/COMMD1 axis was thought to be an important regulator in Cu metabolism maintenance. In this study, the role of XIAP-regulated COMMD1 protein degradation in Pb-induced Cu disorders in brain barrier cells was investigated. Pb exposure significantly increased Cu levels in both cell types, according to atomic absorption technology testing. Western blotting and reverse transcription PCR (RT-PCR) showed that COMMD1 protein levels were significantly increased, whereas XIAP, ATP7A, and ATP7B protein levels were significantly decreased. However, there were no significant effects at the messenger RNA (mRNA) level (XIAP, ATP7A, and ATP7B). Pb-induced Cu accumulation and ATP7B expression were reduced when COMMD1 was knocked down by transient small interfering RNA (siRNA) transfection. In addition, transient plasmid transfection of XIAP before Pb exposure reduced Pb-induced Cu accumulation, increased COMMD1 protein levels, and decreased ATP7B levels. In conclusion, Pb exposure can reduce XIAP protein expression, increase COMMD1 protein levels, and specifically decrease ATP7B protein levels, resulting in Cu accumulation in brain barrier cells.

STAT3 ameliorates cognitive deficits via regulation of NMDAR expression in an Alzheimer's disease animal model.

Background: Abnormal tau accumulation in the brain has a positively correlation with neurodegeneration and memory deterioration, but the mechanism underlying tau-associated synaptic and cognitive impairments remains unclear. Our previous work has found that human full length tau (hTau) accumulation activated signal transducer and activator of transcription-1 (STAT1) to suppress N-methyl-D-aspartate receptors (NMDARs) expression, followed by memory deficits. STAT3 also belongs to STAT protein family and is reported to involve in regulation of synaptic plasticity and cognition. Here, we investigated the role of STAT3 in the cognitive deficits induced by hTau accumulation. Methods:In vitro studies HEK293 cells were used. EMSA, Luciferase reporter assay, and Immunoprecipitation were applied to detect STAT3 activity. In vivo studies, AAV virus were injected into the hippocampal CA3 region of C57 mice. Western blotting, quantitative real-time polymerase chain reaction, and immunofluorescence were applied to examine the level of synaptic proteins. Electrophysiological analysis, behavioral testing and Golgi impregnation were used to determine synaptic plasticity and memory ability recovery after overexpressing STAT3 or non-acetylated STAT1. Results: Our results showed that hTau accumulation acetylated STAT1 to retain STAT3 in the cytoplasm by increasing the binding of STAT1 with STAT3, and thus inactivated STAT3. Overexpressing STAT3 or non-acetylated STAT1 ameliorated hTau-induced synaptic loss and memory deficits by increasing the expression of NMDARs. Conclusions: Taken together, our study indicates that hTau accumulation impaired synaptic plasticity through STAT3 inactivation induced suppression of NMDARs expression, revealing a novel mechanism for hTau-associated synapse and memory deficits.

Maternal Lead Exposure Impairs Offspring Learning and Memory via Decreased GLUT4 Membrane Translocation.

Lead (Pb) can cause a significant neurotoxicity in both adults and children, leading to the impairment to brain function. Pb exposure plays a key role in the impairment of learning and memory through synaptic neurotoxicity, resulting in the cognitive function. Researches have demonstrated that Pb exposure plays an important role in the etiology and pathogenesis of neurodegenerative diseases, such as Alzheimer's disease. However, the underlying mechanisms remain unclear. In the current study, a gestational Pb exposure (GLE) rat model was established to investigate the underlying mechanisms of Pb-induced cognitive impairment. We demonstrated that low-level gestational Pb exposure impaired spatial learning and memory as well as hippocampal synaptic plasticity at postnatal day 30 (PND 30) when the blood concentration of Pb had already recovered to normal levels. Pb exposure induced a decrease in hippocampal glucose metabolism by reducing glucose transporter 4 (GLUT4) levels in the cell membrane through the phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) pathway. In vivo and in vitro GLUT4 over-expression increased the membrane translocation of GLUT4 and glucose uptake, and reversed the Pb-induced impairment to synaptic plasticity and cognition. These findings indicate that Pb exposure impairs synaptic plasticity by reducing the level of GLUT4 in the cell membrane as well as glucose uptake via the PI3K-Akt signaling pathway, demonstrating a novel mechanism for Pb exposure-induced neurotoxicity.

An efficient autometallography approach to localize lead at ultra-structural levels of cultured cells.

Understanding the precise intracellular localization of lead (Pb) is a key in deciphering processes in Pb-induced toxicology. However, it is a great challenge to trace Pb in vitro, especially in cultured cells. We aimed to find an innovative and efficient approach to investigate distribution of Pb in cells and to validate it through determining the subcellular Pb content. We identified its ultra-structural distribution with autometallography under electron microscopy in a choroidal epithelial Z310 cell line. Electron microscopy in combination with energy-dispersive X-ray spectroscope (EDS) was employed to provide further evidence of Pb location. In addition, Pb content was determined in the cytosol, membrane/organelle, nucleus and cytoskeleton fractions with atomic absorption spectroscopy. Pb was found predominantly inside the nuclear membranes and some was distributed in the cytoplasm under low-concentration exposure. Nuclear existence of Pb was verified by EDS under electron microscopy. Once standardized for protein content, Pb percentage in the nucleus fraction reached the highest level (76%). Our results indicate that Pb is accumulated mainly in the nucleus of choroid plexus. This method is sensitive and precise in providing optimal means to study the ultra-structural localization of Pb for in vitro models. In addition, it offers the possibility of localization of other metals in cultured cells. Some procedures may also be adopted to detect target proteins via immunoreactions.

Low-level Gestational Lead Exposure Alters Dendritic Spine Plasticity in the Hippocampus and Reduces Learning and Memory in Rats.

Lead (Pb) is known to impair children's cognitive function. It has been previously shown that developmental Pb exposure alters dendritic spine formation in hippocampal pyramidal neurons. However, the underlying mechanism has not yet been defined. In this study, a low-level gestational Pb exposure (GLE) rat model was employed to investigate the impact of Pb on the spine density of the hippocampal pyramidal neurons and its regulatory mechanism. Pb exposure resulted in impaired performance of the rats in the Morris water maze tasks, and in decreased EPSC amplitudes in hippocampal CA3-CA1 regions. With a 3D reconstruction by the Imaris software, the results from Golgi staining showed that the spine density in the CA1 region was reduced in the Pb-exposed rats in a dose-dependent manner. Decreased spine density was also observed in cultured hippocampal neurons following the Pb treatment. Furthermore, the expression level of NLGN1, a postsynaptic protein that mediates synaptogenesis, was significantly decreased following the Pb exposure both in vivo and in vitro. Up-regulation of NLGN1 in cultured primary neurons partially attenuated the impact of Pb on the spine density. Taken together, our resultssuggest that Pb exposure alters spine plasticity in the developing hippocampus by down-regulating NLGN1 protein levels.

Adult lead exposure increases blood-retinal permeability: A risk factor for retinal vascular disease.

Low-to-moderate level developmental and adult lead exposure produces retinal dysfunction and/or degeneration in humans and experimental animals. Although high level in vivo or in vitro lead disrupts blood-brain-barrier tight junctions and increases its permeability, the blood-retinal-barrier (BRB) has not been examined. There were four overall goals. First, generate environmentally relevant dose-response models of short-term lead exposure in adult rats. Second, assess retinal histology and functional integrity of the BRB. Third, investigate the transmembrane proteins occludin and claudin-5 as targets mediating the increased BRB permeability. Fourth, examine the contribution of the PI3K-Akt signaling pathway as a mechanism underlying increased BRB permeability. Young adult rats were given water, 0.01% or 0.02% lead drinking solutions for six weeks. In control, 0.01% and 0.02% groups the six week mean blood [Pb] were 1, 12.5 and 19µg/dl, respectively. We employed histology, stereology, quantitative image analysis, immunoblots and densitometry, and pharmacology techniques. Major findings were that adult lead exposure produced dose-dependent 1) decreases in outer and inner nuclear layer thickness, 2) increases in BRB permeability, 3) decreases in occludin and claudin-5 expression, 4) increases in pAkt (Ser473), but not pAkt (Thr308), expression, and 5) wortmannin partially or completely blocked the increased BRB permeability and changes in protein expression. These results indicate that lead-induced increases in PI3K-Akt signaling partially underlie the increased BRB permeability and advance our knowledge about lead-induced retinotoxicity. Furthermore, they suggest that environmental and occupational lead exposures are risk factors for increased BRB permeability in diseases such as age-related macular degeneration, diabetes and stroke.

Cellular uptake of lead in the blood-cerebrospinal fluid barrier: Novel roles of Connexin 43 hemichannel and its down-regulations via Erk phosphorylation.

As the structural basis of blood-cerebrospinal fluid barrier (BCB), epithelial cells in the choroid plexus (CP) are targets for lead (Pb). Pb is known to accumulate in the CP; however, the mechanism of Pb uptake in the choroidal epithelial cells remains unknown. Recently, hemichannels of Connexin 43 (Cx43), the most ubiquitously expressed gap junction proteins in the CP, were found to be important pathways for many substances. This study was designed to investigate the roles of Cx43 in Pb uptake in the epithelial cells. Autometallography was used to outline Pb's subcellular location, and the characteristics of Pb transport into CP cells, including concentration- and time-dependence were analyzed by atomic absorption spectroscopy. Knockdown/overexpression of Cx43 with transient siRNA/plasmids transfections before Pb exposure diminished/increased the Pb accumulation. In the Z310 cell-based doxycycline-inducible Cx43 expression cell line (iZCx43), doxycycline induced a significant increase (3-fold) in Pb uptake, corresponding to the increased Cx43 levels. Activation of Cx43 hemichannels by reduced serum conditions caused an increase of Pb concentrations. Cx43-induced Pb uptake was attenuated after blockage of Cx43 hemichannels with its inhibitor, carbenoxolone. Additionally, down-regulation of Cx43 protein levels by Pb exposure paralleled cellular Pb concentrations in the time study. Concomitantly, expressions of phosphor-Src and phosphor-Erk were both significantly increased by Pb. However, inactivation of Erk, not Src pathway, reversed Pb-induced downregulation of Cx43. Taken together, these data establish that Pb can accumulate in the BCB and validate the role of Cx43 hemichannel in Pb uptake and its regulations through Erk phosphorylation.