Involvement of insulin-degrading enzyme in the clearance of beta-amyloid at the blood-CSF barrier: Consequences of lead exposure

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) is characterized by the deposition of beta-amyloid (Aβ) peptides in the brain extracellular matrix, resulting in pathological changes and neurobehavioral deficits. Previous work from this laboratory demonstrated that the choroid plexus (CP) possesses the capacity to remove Aβ from the cerebrospinal fluid (CSF), and exposure to lead (Pb) compromises this function. Since metalloendopeptidase insulin-degrading enzyme (IDE), has been implicated in the metabolism of Aβ, we sought to investigate whether accumulation of Aβ following Pb exposure was due to the effect of Pb on IDE.</p> <p>Methods</p> <p>Rats were injected with a single dose of Pb acetate or an equivalent concentration of Na-acetate; CP tissues were processed to detect the location of IDE by immunohistochemistry. For <it>in vitro </it>studies, choroidal epithelial Z310 cells were treated with Pb for 24 h in the presence or absence of a known IDE inhibitor, N-ethylmaleimide (NEM) to assess IDE enzymatic activity and subsequent metabolic clearance of Aβ. Additionally, the expression of IDE mRNA and protein were determined using real time PCR and western blots respectively.</p> <p>Results</p> <p>Immunohistochemistry and confocal imaging revealed the presence of IDE towards the apical surface of the CP tissue with no visible alteration in either its intensity or location following Pb exposure. There was no significant difference in the expressions of either IDE mRNA or protein following Pb exposure compared to controls either in CP tissues or in Z310 cells. However, our findings revealed a significant decrease in the IDE activity following Pb exposure; this inhibition was similar to that seen in the cells treated with NEM alone. Interestingly, treatment with Pb or NEM alone significantly increased the levels of intracellular Aβ, and a greater accumulation of Aβ was seen when the cells were exposed to a combination of both.</p> <p>Conclusion</p> <p>These data suggest that Pb exposure inhibits IDE activity but does not affect its expression in the CP. This, in turn, leads to a disrupted metabolism of Aβ resulting in its accumulation at the blood-CSF barrier.</p

Acute and developmental behavioral effects of flame retardants and related chemicals in zebrafish

As polybrominated diphenyl ethers are phased out, numerous compounds are emerging as potential replacement flame retardants for use in consumer and electronic products. Little is known, however, about the neurobehavioral toxicity of these replacements. This study evaluated the neurobehavioral effects of acute or developmental exposure to t-butylphenyl diphenyl phosphate (BPDP), 2-ethylhexyl diphenyl phosphate (EHDP), isodecyl diphenyl phosphate (IDDP), isopropylated phenyl phosphate (IPP), tricresyl phosphate (TMPP; also abbreviated TCP), triphenyl phosphate (TPHP; also abbreviated TPP), tetrabromobisphenol A (TBBPA), tris (2-chloroethyl) phosphate (TCEP), tris (1,3-dichloroisopropyl) phosphate (TDCIPP; also abbreviated TDCPP), tri-o-cresyl phosphate (TOCP), and 2,2-,4,4′-tetrabromodiphenyl ether (BDE-47) in zebrafish (Danio rerio) larvae. Larvae (n ≈ 24 per dose per compound) were exposed to test compounds (0.4–120 µM) at sub-teratogenic concentrations either developmentally or acutely, and locomotor activity was assessed at 6 days post fertilization. When given developmentally, all chemicals except BPDP, IDDP and TBBPA produced behavioral effects. When given acutely, all chemicals produced behavioral effects, with TPHP, TBBPA, EHDP, IPP, and BPDP eliciting the most effects at the most concentrations. The results indicate that these replacement flame retardants may have developmental or pharmacological effects on the vertebrate nervous system

Alteration of β-amyloid clearance by the blood - cerebrospinal fluid barrier following lead exposure

Lead (Pb) toxicity remains a major public health concern due to its continued use in the modern industry. Recent evidence demonstrates an association between occupational Pb exposure and cognitive dysfunction. One of the major pathological hallmarks of Alzheimer’s disease (AD) is the extensive presence of beta-amyloid (Aβ) in the brain and cerebrospinal fluid (CSF). Autopsies of brains from AD patients reveal distinct Aβ stains in the choroid plexus (CP), a region where the blood-cerebrospinal fluid barrier (BCB) is located. The CP, in addition to being involved in the uptake and metabolism of Aβ from the CSF, has shown to be a target for Pb accumulation in the brain. Currently there is no known link between Pb exposure and Aβ regulation at the BCB. Hence, this study was designed to investigate a possible link between Pb exposure and alteration in Aβ transport and clearance by the CP by potentially influencing: (i) alterations in the expression and/or subcellular localization of Aβ efflux transporter, Lipoprotein Receptor Protein 1 (LRP1), and/or (ii) alterations in the expression, localization and/or activitiy of Aβ degrading enzyme, Insulin Degrading Enzyme (IDE). Results from laser scanning cytometry studies revealed a significant increase in intracellular Aβ levels in Pb-exposed rats relative to controls (p\u3c0.001). In vitro ELISA studies in choroid plexus epithelial (Z310) cells demonstrated a dose-dependent and time-related increase in Aβ accumulation following 24 h or 48 h of Pb exposure. To explore a mechanism for the above finding, the involvement of Aβ transporter, LRP1 was assessed. Results from real time RT-PCR and Western blot analysis revealed a significant decrease by 35% (p\u3c0.05) in mRNA and 31.8% (p\u3c0.05) in protein expression in the CP of rats exposed to Pb compared to controls. In vitro studies revealed a 33.1% and 33.4% decrease in the protein expression of LRP1 (p\u3c0.05) following Pb exposure (10 μM) for 24 h or 48 h respectively. Knocking down LRP1 by siRNA resulted in an increase in the cellular accumulation of Aβ, from 31% to 72% (p\u3c0.05). A distinct subcellular relocalization of LRP1 was observed from the cytosol in control tissues to the apical surface and microvilli following Pb exposure. Double immunostaining of LRP1 with PKC-δ revealed a co-localization of both in the cytosol of controls with a distinct relocalization following Pb treatment. Interestingly, pre-incubation with PKC-δ inhibitor rottlerin, abolished the Pb-induced relocalization of LRP1 as well as the Pb-induced Aβ accumulation. Next, we assessed the involvement of IDE, an Aβ degrading enzyme. Results from the IDE enzymatic activity assay revealed a significant decrease in activity in cells exposed to Pb compared to controls with a corresponding increase in Aβ accumulation. There was no visible relocalization of IDE either in the CP tissues or in Z310 cells following Pb exposure, as indicated by immunohistochemistry and confocal imaging. Additionally, there was no significant difference in IDE mRNA or protein expression as determined by real time RT-PCR and Western blot analysis. Based on our findings, we conclude that Pb exposure results in Aβ dysregulation at the BCB by a combination of mechanisms involving LRP1 and IDE. These alterations in Aβ clearance in the CP may have significant implications for the subsequent build up of Aβ in surrounding brain regions like the hippocampus and cortex, and may potentially result in exacerbating AD-like pathology

International regulatory and scientific effort for improved developmental neurotoxicity testing

The Organisation for Economic Co-operation and Development (OECD) coordinates international efforts to enhance developmental neurotoxicity (DNT) testing. In most regulatory sectors, including the ones dealing with pesticides and industrial chemicals registration, historical use of the in vivo DNT test guideline has been limited. Current challenges include a lack of DNT data for a number of chemicals, lack of mechanistic information, and difficulty in interpreting results. A series of workshops in the last decade has paved the way for a consensus among stakeholders that there is need for a DNT testing battery that relies on in vitro methods and is complemented by alternative species assays. Preferably, a battery of in vitro and alternative assays should be anchored towards mechanistic relevance for applying an integrated approach for testing and assessment (IATA) framework. Specific activities have been initiated to facilitate this OECD project: the collation of available DNT in vitro methods and their scoring for readiness; the selection of these methods to form a DNT testing battery; the generation of a reference set of chemicals that will be tested using the battery; the case studies exemplifying how DNT in vitro data can be interpreted; and the development of an OECD guidance document. This manuscript highlights these international efforts and activities.JRC.F.3-Chemicals Safety and Alternative Method

Endpoint (quantity type) data from the neurotoxicity assay

Endpoints include L1_distmoved, D1_distmoved, L2_distmoved, , and D2_distmoved. Responses include both normalized and non-normalized

Raw data from the neurotoxicity assay

The distance moved (mm) per min dat

Endpoint data from developmental toxicity assay

The endpoints include percent_affected, percent_malformed, n_dead per well, n_malformed per wel

Zebrafish data transfer

Examples were included to demonstrate how to load the data file