BACE-1 inhibition prevents the γ-secretase inhibitor evoked Aβ rise in human neuroblastoma SH-SY5Y cells

<p>Abstract</p> <p>Background</p> <p>Accumulation of amyloid β-peptide (Aβ) in the plaques is one of the major pathological features in Alzheimer's disease (AD). Sequential cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE-1) and γ-secretase results in the formation of Aβ peptides. Preventing Aβ formation is believed to attenuate AD progression and BACE-1 and γ-secretase are thus attractive targets for AD drug development.</p> <p>Methods</p> <p>Combining BACE-1 and γ-secretase inhibition on Aβ secretion from human neuroblastoma SH-SY5Y cells was evaluated in this study. Secreted Aβ40 and Aβ42 levels were measured from SH-SY5Y cells stably transfected with APPwt or APPswe genes. A selective BACE inhibitor and the γ-secretase inhibitor LY450139 (semagacestat) were used to inhibit respective secretase.</p> <p>Results</p> <p>LY450139 increased Aβ40 and Aβ42 secretion from SH-SY5Y APPwt cells at low concentrations (by 60% at 3 nM) followed by subsequent inhibition at higher concentrations (IC₅₀90 nM). Washout studies showed that the Aβ increase evoked by 3 nM LY450139 was not due to enhanced cleavage following substrate accumulation but rather to activation of Aβ formation. By contrast, LY450139 inhibited Aβ formation from SH-SY5Y APPswe in a monophasic manner (IC₅₀18 nM). The BACE inhibitor <it>per se </it>inhibited Aβ secretion from both SH-SY5Y APPwt and SH-SY5Y APPswe cells with IC₅₀s ranging between 7 - 18 nM and also prevented the increased Aβ secretion evoked by 3 nM LY450139. Combining the BACE inhibitor with higher inhibitory concentrations of LY450139 failed to demonstrate any clear additive or synergistic effects.</p> <p>Conclusion</p> <p>BACE-1 inhibition attenuates the Aβ increase evoked by LY450139 while not providing any obvious synergistic effects on LY450139-mediated inhibition.</p

Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening

<p>Abstract</p> <p>Background</p> <p>Deregulated activation of cyclin-dependent kinase-5 (Cdk5) is implicated in neurodegenerative disorders such as Alzheimer's disease. One of the restricting factors for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized cellular in vitro assay systems.</p> <p>Methods</p> <p>HEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we utilized an antibody that recognizes phospho serine in a consensus motif for Cdk substrates.</p> <p>Results</p> <p>Western blot analysis of transfected cells detected a 200 kDa band that was identified, by mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B). Phosphorylation of NMHC-B was evident only in cells that were double transfected with Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors. Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y cell line.</p> <p>Conclusion</p> <p>A novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.</p

Активность микрофлоры как показатель токсичности морских донных отложений шельфовой зоны Черного моря и Керченского пролива

Изучена потенциальная активность донной микрофлоры в местах утечки остатков химических токсикантов, затопленных в период Второй Мировой войны ХХ в. Отмечены особенности восстановления жизнедеятельности микрофлоры при различных уровнях загрязнения донных отложений мышьяком и хлорированными органическими сульфидами. Полученные результаты перспективно использовать при оценке экологического состояния донных отложений в загрязненных прибрежных акваториях

In vitro modelling of tau phosphorylating kinases : emphasis of Cdk5

The main hallmarks of Alzheimer’s disease (AD) are extracellular deposits of betaamyloid (Aβ) and intracellular neurofibrillary tangles (NFT) composed of highly phosphorylated tau protein. Abnormal hyperphosphorylation of tau is the most deleterious step in NFT formation making the use of kinase inhibitors an attractive treatment possibility in AD. To enable development and screening of selective kinase inhibitors, well-characterized cellular assays are essential. In this thesis, different cell culture systems were investigated as in vitro models for tau phosphorylating kinases, with an emphasis on cyclin-dependent kinase-5 (Cdk5). In paper I, differentiated SH-SY5Y cell line was investigated as a model for tau phosphorylation. Sequential differentiation of SH-SY5Y cells with retinoic acid and brainderived neurotrophic factor induced a prominent increase in the content and the phosphorylation state of tau. Of the investigated kinases, glycogen synthase kinase 3β (Gsk3β) contributed most to tau phosphorylation whereas Cdk5 made a minor contribution. Lithium, a GSK3β-inhibitor, reproducibly inhibited tau phosphorylation in a wide concentration range indicating that this model can be used to screen for GSK3β inhibitors. In paper II, differentiated SH-SY5Y cells were treated with neurotoxic stimuli or transfected with p25 in order to activate Cdk5. Glutamate increased Cdk5 and p35 protein levels thereby elevating Cdk5 activity and tau phosphorylation. When p25 was transfected to the cells, increased tau phosphorylation was achieved but could not be reduced with the Cdk5 inhibitor Roscovitine. This is possibly through activation of ERK1/2, another tau phosphorylating kinase, detected in Roscovitine treated cells. An additional finding of this study was degradation of p25 via proteasome in cells treated with Cdk5 inhibitors. In paper III, investigation of Aβ treated hippocampal organotypic cultures revealed increased tau phosphorylation at the Ser396 epitope probably through activation of Gsk3β whereas Cdk5 involvement was not detected. In paper IV, alternative Cdk5 substrates were looked for in Cdk5/p25 transfected HEK293 cells. A non-muscle myosin heavy chain, type B (NMHC-B), was identified as a novel Cdk5 substrate. Only Cdk5 phosphorylates NMHC-B in HEK293 cells and its phosphorylation was concentration-dependently inhibited with Cdk5 inhibitors. A new screening system for Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out. Many kinases, some with reciprocal interactions, are involved in tau phosphorylation in differentiated SH-SY5Y cells complicating its use as a model for Cdk5 mediated tau phosphorylation. The assay with NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells is, however, very specific and sensitive and allows validation of compounds designed to inhibit Cdk5