APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer''s disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis.SIGNIFICANCE STATEMENT Defective protein synthesis has been shown as an early defect in familial Alzheimer''s disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.     

SUPERFACT: A Model Fuel for Studying the Evolution of the Microstructure of Spent Nuclear Fuel during Storage/Disposal

The transmutation of minor actinides (in particular, Np and Am), which are among the main contributors to spent fuel α-radiotoxicity, was studied in the SUPERFACT irradiation. Several types of transmutation UO₂-based fuels were produced, differing by their minor actinide content (²⁴¹Am, ²³⁷Np, Pu), and irradiated in the Phénix fast reactor. Due to the high content in rather short-lived alpha-decaying actinides, both the archive, but also the irradiated fuels, cumulated an alpha dose during a laboratory time scale, which is comparable to that of standard LWR fuels during centuries/millenaries of storage. Transmission Electron Microscopy was performed to assess the evolution of the microstructure of the SUPERFACT archive and irradiated fuel. This was compared to conventional irradiated spent fuel (i.e., after years of storage) and to other ²³⁸Pu-doped UO₂ for which the equivalent storage time would span over centuries. It could be shown that the microstructure of these fluorites does not degrade significantly from low to very high alpha-damage doses, and that helium bubbles precipitate.     

Heat-shock protein expression in cisplatin-sensitive and -resistant human tumor cells

It has been suggested that the expression of certain heat-shock proteins (HSPs) may be prognostic markers in several tumor types. Since HSPs may be involved in determining cellular sensitivity to chemotherapeutic drugs, the possible relation between HSP expression and cisplatin (cDDP) sensitivity was studied. Three human germ-cell tumor cell lines, 1 human small-cell lung carcinoma (SCLC) cell line and 3 human colon carcinoma cell lines were used as a model for differences in intrinsic cDDP sensitivity. The constitutive expression of a panel of HSPs was studied by immunoblotting. No correlation was found between expression of HSP90, HSP73, HSP72, HSP60 and HSP27 and the extent of intrinsic cDDP sensitivity when all cell lines studied were considered. However, for the 3 cell lines derived from germ-cell carcinomas, HSP27 expression was inversely related to cDDP sensitivity, i.e. decreased HSP27 levels were associated with decreased sensitivity. Constitutive HSP expression was also studied in 2 sets of human cell lines with in vitro acquired cDDP resistance. In both resistant cell lines, decreased expression of HSP27 (as determined by Western blotting) was found as compared to the sensitive parent cell lines. Thus, acquired resistance to cDDP was also accompanied by decreased HSP27 expression. Interestingly, when basal HSP27 mRNA levels were measured in the SCLC cell line (GLC₄) and its subline with acquired resistance (GLC₄-cDDP), no significant differences were detected. Continuous cDDP incubation increased HSP27 levels and induced HSP27 phosphorylation in GLC₄ cells, but not in the resistant subline. Thus, although no general relationships between HSP expression and cDDP sensitivity are apparent, high HSP27 expression in vitro relates to high sensitivity to cDDP treatment in some tumor types. This is in accordance with reported clinical data on high HSP27 levels in tumors correlating with good prognosis. © 1996 Wiley-Liss, Inc.     

Cigarette smoke targets glutaredoxin 1, increasing s-glutathionylation and epithelial cell death

It is established that cigarette smoke (CS) causes irreversible oxidations in lung epithelial cells, and can lead to their death. However, its impact on reversible and physiologically relevant redox-dependent protein modifications remains to be investigated. Glutathione is an important antioxidant against inhaled reactive oxygen species as a direct scavenger, but it can also covalently bind protein thiols upon mild oxidative stress to protect them against irreversible oxidation. This posttranslational modification, known as S-glutathionylation, can be reversed under physiological conditions by the enzyme, glutaredoxin 1 (Grx1). The aim of this study was to investigate if CS modifies Grx1, and if this impacts on protein S-glutathionylation and epithelial cell death. Upon exposure of alveolar epithelial cells to CS extract (CSE), a decrease in Grx1 mRNA and protein expression was observed, in conjunction with decreased activity and increased protein S-glutathionylation. Using mass spectrometry, irreversible oxidation of recombinant Grx1 by CSE and acrolein was demonstrated, which was associated with attenuated enzyme activity. Furthermore, carbonylation of Grx1 in epithelial cells after exposure to CSE was shown. Overexpression of Grx1 attenuated CSE-induced increases in protein S-glutathionylation and increased survival. Conversely, primary tracheal epithelial cells of mice lacking Grx1 were more sensitive to CS-induced cell death, with corresponding increases in protein S-glutathionylation. These results show that CS can modulate Grx1, not only at the expression level, but can also directly modify Grx1 itself, decreasing its activity. These findings demonstrate a role for the Grx1/S-glutathionylation redox system in CS-induced lung epithelial cell death.     

Development of a tapping device: a new needle insertion method for prostate brachytherapy

The purpose of this study is to develop and test a tapping device for needle insertion for prostate brachytherapy. This device will tap the needle into the prostate with a certain, well-defined, amount of momentum, instead of the currently used method of pushing the needle. Because of the high needle insertion velocity, we expect prostate motion and deformation to be less compared to current methods. We measured the momentum that is applied when manually tapping the needle into the prostate and found a mean momentum of 0.50 +/- 0.07 N s. The tapping device is pneumatically driven and we found that the delivered momentum increased linearly with the applied air pressure. The efficacy of the tapping device was tested on a piece of beef, placed on a freely moving and rotating platform. A significant correlation was found between the applied pressure and the rotation and displacement of the beef. Displacements and rotations were minimal for the highest pressure (4 bar) and amounted to only 2 mm and 6 degrees, respectively. Higher air pressures will further reduce displacements and rotations.     

Temperature‐induced tissue susceptibility changes lead to significant temperature errors in PRFS‐based MR thermometry during thermal interventions

Proton resonance frequency shift‐based MR thermometry (MRT) is hampered by temporal magnetic field changes. Temporal changes in the magnetic susceptibility distribution lead to nonlocal field changes and are, therefore, a possible source of errors. The magnetic volume susceptibility of tissue is temperature dependent. For water‐like tissues, this dependency is in the order of 0.002 ppm/°C. For fat, it is in the same order of magnitude as the temperature dependence of the proton electron screening constant of water (0.01 ppm/°C). For this reason, proton resonance frequency shift‐based MR thermometry in fatty tissues, like the human breast, is expected to be prone to errors. We aimed to quantify the influence of the temperature dependence of the susceptibility on proton resonance frequency shift‐based MR thermometry. Heating experiments were performed in a controlled phantom set‐up to show the impact of temperature‐induced susceptibility changes on actual proton resonance frequency shift‐based temperature maps. To study the implications for a clinical case, simulations were performed in a 3D breast model. Temperature errors were quantified by computation of magnetic field changes in the glandular tissue, resulting from susceptibility changes in a thermally heated region. The results of the experiments and simulations showed that the temperature‐induced susceptibility changes of water and fat lead to significant errors in proton resonance frequency shift‐based MR thermometry. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Phase I and pharmacological study of the broad-spectrum tyrosine kinase inhibitor JNJ-26483327 in patients with advanced solid tumours

Background:

JNJ-26483327 is an oral, potent, multi-targeted tyrosine kinase inhibitor, inhibiting kinases of epidermal growth factor receptor (EGFR)-1, -2 and -4, rearranged during transfection (RET) receptor, vascular endothelial growth factor receptor (VEGFR)-3 and Src family (Lyn, Fyn, Yes) at low nanomolar concentrations. This phase I, accelerated titration study assessed maximum tolerated dose, safety, pharmacokinetics and pharmacodynamic effects of JNJ-26483327.

Methods:

Nineteen patients with advanced cancers received JNJ-26483327 continuous twice daily (BID) in escalating dose cohorts ranging from 100 to 2100 mg. Pharmacodynamic effects were assessed in paired skin biopsies and blood.

Results:

JNJ-26483327 was well tolerated in doses up to 1500 mg BID, with target-inhibition-related toxicity such as diarrhoea and skin rash, and other common reported toxicities being nausea, vomiting, anorexia and fatigue. At 2100 mg, two episodes of dose-limiting toxicity were observed, consisting of grade 3 anorexia and a combination of grade 3 anorexia and fatigue, respectively. Pharmacokinetics were dose proportional up to 1500 mg in which plasma levels were obtained showing anti-tumour activity in xenograft mouse models. Pharmacodynamic analysis did not show a substantial effect on expression of Ki-67, p27^kip1, phosphorylated mitogen-activated protein kinase, phosphorylated Akt and EGFR, and serum levels of sVEGFR-2, VEGF-C and VEGF-D remained unchanged. Stable disease was noted in six patients (32%).

Conclusion:

JNJ-26483327 is well tolerated and shows a predictable pharmacokinetic profile; the recommended dose for further studies is 1500 mg BID.     

Dietary acrylamide intake and estrogen and progesterone receptor-defined postmenopausal breast cancer risk

Acrylamide, a potential human carcinogen, has been discovered in a variety of heat-treated carbohydrate-rich food products. Previously, dietary acrylamide intake was shown to be associated with endocrine-related cancers in humans. We assessed the association between dietary acrylamide intake and risk of postmenopausal breast cancer stratified by estrogen and progesterone receptor status. This study was embedded within the Netherlands Cohort Study on diet and cancer, which was initiated in 1986 enrolling 62,573 women aged 55–69 years at baseline. After 13.3 years of follow-up, 2225 incident breast cancer cases were ascertained, with hormone receptor status information for 43%. Cox proportional hazards analysis was applied to determine hazard ratios in quintiles of dietary acrylamide intake stratifying on estrogen receptor (ER) and progesterone receptor (PR) and smoking status. No association was observed for overall breast cancer or receptor-negative breast cancer risk, irrespective of smoking status. A statistically non-significantly increased risk of ER positive, PR positive and joint receptor-positive breast cancer was found in never-smoking women. The multivariable-adjusted hazard ratios were 1.31 (95% CI: 0.87–1.97, P _trend = 0.26) for ER+, 1.47 (0.86–2.51, P _trend = 0.14) for PR+, and 1.43 (0.83–2.46, P _trend = 0.16) for ER+PR+, when comparing women in the highest quintile of acrylamide intake (median 36.8 μg/day) to women in the lowest (median 9.5 μg/day). This study showed some indications of a positive association between dietary acrylamide intake and receptor-positive breast cancer risk in postmenopausal never-smoking women. Further studies are needed to confirm or refute our observations.     

Radiation induced cell loss in rat submandibular gland and its relation to gland function

Purpose : To understand early and late radiation-induced loss of function of the submandibular gland, changes in cell number were documented and correlated with data on gland function. Modulation of the radiation effect by sialogogues was used to investigate possible mechanisms of action. Materials and methods : Rats were irradiated with a single dose of 15 Gy of X-rays after pre-treatment with either saline, the muscarinic receptor agonists methacholine or pilocarpine, the adrenergic receptor agonist phenylephrine or methacholine plus phenylephrine. Before and 1-240 days after irradiation, submandibular saliva flow rate was measured. At the same time points and from comparable animals submandibular glands were carefully extirpated, weighed and prepared for light microscopic examination. Results : Soon after irradiation (<30 days) no significant loss of cells was observed, whereas the gland function was severely compromized. Sialogogue pre-treatment attenuated the radiation-induced loss of gland function. At later intervals a considerable loss of acinar cells and to a lesser extent loss of granular convoluted tubule cells were observed. Gland function subsequently declined slowly. Pre-treatment with sialogogues gave transient protection against cell loss and loss of gland function. Conclusions : The lack of cell loss observed soon after irradiation indicates that the observed reduction in gland function was caused by a compromised functioning of the acini. The later loss of cells is probably due to death of cells that normally proliferate, leading to a further reduced secretory capacity. Protection of gland morphology and function by sialogogues at later times must therefore involve resistance of progenitor cells to radiation-induced cell death.