{"id":2576,"date":"2020-02-05T12:32:13","date_gmt":"2020-02-05T12:32:13","guid":{"rendered":"https:\/\/a031212db6.nxcli.net\/in-vitro-services\/alzheimers-disease-in-vitro-models\/"},"modified":"2023-11-29T18:59:13","modified_gmt":"2023-11-30T00:59:13","slug":"alzheimers-disease-in-vitro-models","status":"publish","type":"page","link":"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/kr\/in-vitro-services\/alzheimers-disease-in-vitro-models\/","title":{"rendered":"\uc54c\uce20\ud558\uc774\uba38\ubcd1 \uccb4\uc678 \ubaa8\ub378"},"content":{"rendered":"<section class=\"gb-container gb-container-0d0de91c\">\n<div class=\"gb-container gb-container-b147caa0\">\n<div class=\"gb-grid-wrapper gb-grid-wrapper-a97e7cf0\">\n<div class=\"gb-grid-column gb-grid-column-649baef0\"><div class=\"gb-container gb-container-649baef0\">\n\n<p>Alzheimer\u2019s disease (AD) is one of the most devastating neurodegenerative diseases. Aggregation of amyloid-beta peptide (A\u03b2) into cytotoxic oligomers and fibrills is one of the major hallmarks in AD. These pathological depositions in the brain are thought to be one of the main causes for the observed progressive cognitive decline in AD patients.<\/p>\n\n\n\n<p>Interfering with A\u03b2 aggregation is an inevitable strategy in the development of novel therapeutic approaches. Reliable&nbsp;<em>in vitro<\/em>&nbsp;models are needed, which are capable of showing direct effects of compounds on A\u03b2 oligomer formation and thus, a beneficial impact on cell viability.<\/p>\n\n\n<div class=\"gb-container gb-container-f0fb5caf\">\n<div class=\"gb-container gb-container-17fa4bc8 gb-accordion\">\n<div class=\"gb-container gb-container-ac3c99c3 gb-accordion__item\" data-transition=\"slide\">\n\n<button class=\"gb-button gb-button-8568b098 gb-accordion__toggle\"><span class=\"gb-button-text\"><strong>A\u03b2 \uc720\ub3c4\uc131 \ub3c5\uc131<\/strong><\/span><span class=\"gb-icon\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon\"><path d=\"M416 208H272V64c0-17.67-14.33-32-32-32h-32c-17.67 0-32 14.33-32 32v144H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h144v144c0 17.67 14.33 32 32 32h32c17.67 0 32-14.33 32-32V304h144c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon-open\"><path d=\"M416 208H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h384c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><\/span><\/button>\n\n\n<div class=\"gb-accordion__content\"><div class=\"gb-container gb-container-d2afecd9\">\n\n<p>QPS Neuropharmacology provides fast and reproducible screening assays to investigate if your developmental compounds are able to protect against cytotoxic effects of aggregated A\u03b21-42. Various&nbsp;<em>in vitro<\/em>&nbsp;models from primary neurons to several cell lines are available and described on the following pages.<\/p>\n\n\n\n<h5 class=\"gb-headline gb-headline-4844e635 gb-headline-text\">Primary rat and mouse neurons<\/h5>\n\n\n\n<p>The hippocampus, a brain area critical for learning and memory, is especially vulnerable to damage at early stages of Alzheimer\u2019s disease (AD). Primary rat\/mouse hippocampal neurons are phenotypically closer to adult neurons as they have extended a dense network of neuronal processes.<\/p>\n\n\n\n<figure class=\"gb-block-image gb-block-image-305f77c2\"><img loading=\"lazy\" decoding=\"async\" width=\"355\" height=\"343\" class=\"gb-image gb-image-305f77c2\" src=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_rat.png\" alt=\"Abeta-toxicity_rat\" title=\"Abeta-toxicity_rat\" srcset=\"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_rat.png 355w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_rat-300x290.png 300w\" sizes=\"(max-width: 355px) 100vw, 355px\" \/><\/figure>\n\n\n\n<p><strong>Figure: Neuroprotective effects of RI on A\u03b21-42 induced toxicity in primary rat hippocampal neurons.<\/strong>&nbsp;Vehicle control, aggregated A\u03b21-42 [1\u00b5M, 48h] or Reference item (RI) with aggregated A\u03b21-42 were applied to primary rat hippocampal neurons. After 144h, cell viability was determined according to MTT assay. RI rescued cell viability and protected against toxic effects of A\u03b21-42.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h5 class=\"gb-headline gb-headline-5b26c080 gb-headline-text\">Primary chicken neurons<\/h5>\n\n\n\n<p>Primary chicken neurons secrete endogenous wildtype A\u03b2 peptides. Both human and chick A\u03b242 are identical in sequence.<\/p>\n\n\n\n<p>Thus, primary chicken neurons are a suitable&nbsp;<em>in vitro<\/em>&nbsp;model for evaluating protective effects of your compounds on A\u03b2 induced neurotoxicity.<\/p>\n\n\n\n<figure class=\"gb-block-image gb-block-image-303a9068\"><img loading=\"lazy\" decoding=\"async\" width=\"809\" height=\"362\" class=\"gb-image gb-image-303a9068\" src=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_chicken.png\" alt=\"Abeta-toxicity_chicken\" title=\"Abeta-toxicity_chicken\" srcset=\"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_chicken.png 809w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_chicken-300x134.png 300w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_chicken-768x344.png 768w\" sizes=\"(max-width: 809px) 100vw, 809px\" \/><\/figure>\n\n\n\n<p><strong>Figure: Neuroprotective effects of compounds on A\u03b21-42 induced toxicity in primary chicken neurons.<\/strong>&nbsp;(A) Compounds were co-aggregated with A\u03b21-42 for 48h and applied to primary chicken neurons on DIV6. After 96h, cell viability was determined according to MTT assay. Co-aggregation of compound Y and Z or reference item (RI) with A\u03b21-42 rescued cell viability and protected against toxic effects of A\u03b21-42. (B) Images illustrate neuroprotective effects of RI when co-aggregated with A\u03b21-42 (c) in comparison to cells treated with aggregated A\u03b21-42 only (b) or control cells (a).<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h5 class=\"gb-headline gb-headline-c452f1ca gb-headline-text\">SH-SY5Y cells<\/h5>\n\n\n\n<p>SH-SY5Y cells is a human derived neuroblastoma cell line and has become a popular&nbsp;<em>in vitro model<\/em>&nbsp;for neurodegenerative diseases.<\/p>\n\n\n\n<figure class=\"gb-block-image gb-block-image-4d825d74\"><img loading=\"lazy\" decoding=\"async\" width=\"453\" height=\"343\" class=\"gb-image gb-image-4d825d74\" src=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_SHSY5Y.png\" alt=\"Abeta-toxicity_SHSY5Y\" title=\"Abeta-toxicity_SHSY5Y\" srcset=\"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_SHSY5Y.png 453w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-toxicity_SHSY5Y-300x227.png 300w\" sizes=\"(max-width: 453px) 100vw, 453px\" \/><\/figure>\n\n\n\n<p><strong>Figure: Evaluation of neuroprotective effects of compounds on A\u03b2 induced toxicity in SH-SY5Y cells.<\/strong>&nbsp;Compounds were applied together with aggregated A\u03b21-42 (1 \u00b5M) to SH-SY5Y cells for 96h. Cell viability was determined according to MTT assay. Reference item and Compound X (at 0.1 and 1\u00b5M) were able to protect against toxic effects of A\u03b21-42.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h5 class=\"gb-headline gb-headline-a8811857 gb-headline-text\">More cell lines<\/h5>\n\n\n\n<p><strong>A\u03b21-42 toxicity assay is also available in:<\/strong><\/p>\n\n\n\n<ul class=\"list__bullets\">\n<li>ARPE-19 cells (human Retina Pigment Epithelial cell line)<\/li>\n\n\n\n<li>PD cells (human fibroblasts from a PD patient)<\/li>\n\n\n\n<li>FRDA cells (human fibroblasts from a Friedreich Ataxia patient)<\/li>\n\n\n\n<li>LHON cells (human fibroblasts from a Leber\u2018s hereditary Optic Neuropathy)<\/li>\n<\/ul>\n\n<\/div><\/div>\n<\/div>\n\n<div class=\"gb-container gb-container-9304f40f gb-accordion__item\" data-transition=\"slide\">\n\n<button class=\"gb-button gb-button-2df9c9a2 gb-accordion__toggle\"><span class=\"gb-button-text\"><strong>A\u03b2 \ud3a9\ud0c0\uc774\ub4dc \ud615\uc131<\/strong> <strong>\ub610\ub294 \uc138\ud06c\ub808\ud0c0\uc81c \uc5b5\uc81c\uc778\uc790 \uc2a4\ud06c\ub9ac\ub2dd<\/strong><\/span><span class=\"gb-icon\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon\"><path d=\"M416 208H272V64c0-17.67-14.33-32-32-32h-32c-17.67 0-32 14.33-32 32v144H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h144v144c0 17.67 14.33 32 32 32h32c17.67 0 32-14.33 32-32V304h144c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon-open\"><path d=\"M416 208H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h384c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><\/span><\/button>\n\n\n<div class=\"gb-accordion__content\"><div class=\"gb-container gb-container-cb74f6ce\">\n\n<p>Generation of A\u03b2 species is a hallmark of Alzheimer\u2019s Disease. QPS Neuropharmacology provides several cell culture models to identify pharmacological agents that interfere with the cleavage of APP by analyzing the generation of different A\u03b2 and sAPP species by immunosorbent assay.<\/p>\n\n\n\n<ul class=\"list__bullets\">\n<li>Primary chicken telencephalic neurons secrete endogenous wildtype A\u03b2 peptides, including A\u03b238, A\u03b240 and A\u03b242. Both human and chick A\u03b242 are identical in sequence. Therefore, this model is suitable for studying effects on processing of endogenous wildtype APP in primary neurons.<\/li>\n\n\n\n<li>Primary rat\/mouse hippocampal neurons are phenotypically closer to adult neurons as they have extended a dense network of neuronal processes.<\/li>\n\n\n\n<li>H4 neuroglioma cells overexpressing human APPK595N\/M596L, the Swedish double mutation, enable to analyze compounds for their effects on the human mutant APP profile.<\/li>\n<\/ul>\n\n\n\n<figure class=\"gb-block-image gb-block-image-978c0c6c\"><img loading=\"lazy\" decoding=\"async\" width=\"757\" height=\"560\" class=\"gb-image gb-image-978c0c6c\" src=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-peptide-profile.png\" alt=\"Abeta-peptide-profile\" title=\"Abeta-peptide-profile\" srcset=\"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-peptide-profile.png 757w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Abeta-peptide-profile-300x222.png 300w\" sizes=\"(max-width: 757px) 100vw, 757px\" \/><\/figure>\n\n\n\n<p><strong>Figure: A\u03b2 peptide profile in primary chicken neurons and APPK595N\/M596L overexpressing cells.<\/strong><\/p>\n\n\n\n<p>(A) H4 cells overexpressing human APPK595N\/M596L generated more A\u03b238, 40, and 42 peptides in 24h than primary chicken neurons determined by an immunosorbent assay. In both, A\u03b240 was the predominant A\u03b2 peptide. (B) The \u03b3-secretase inhibitor DAPT reduced A\u03b2 formation in primary chicken neurons and in H4-hAPPK595N\/M596L. In the H4 cell line, A\u03b2 levels started to decrease at a concentration of 50nM in H4 cells and at 100 nM DAPT in chicken neurons after 24h treatment.<\/p>\n\n\n\n<p><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC3041911\/\" rel=\"noreferrer noopener\" target=\"_blank\">Czvitkovich et al, J Mol Neurosci. 2011; 43(3): 257\u2013267.<\/a><\/p>\n\n<\/div><\/div>\n<\/div>\n\n<div class=\"gb-container gb-container-c89f2ebc gb-accordion__item\" data-transition=\"slide\">\n\n<button class=\"gb-button gb-button-cff07759 gb-accordion__toggle\"><span class=\"gb-button-text\">A\u03b2 \uc751\uc9d1 \uacb0\uc815<\/span><span class=\"gb-icon\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon\"><path d=\"M416 208H272V64c0-17.67-14.33-32-32-32h-32c-17.67 0-32 14.33-32 32v144H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h144v144c0 17.67 14.33 32 32 32h32c17.67 0 32-14.33 32-32V304h144c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon-open\"><path d=\"M416 208H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h384c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><\/span><\/button>\n\n\n<div class=\"gb-accordion__content\"><div class=\"gb-container gb-container-db6213ef\">\n\n<p>The pathological aggregation of amyloid-beta peptides (A\u03b2) is one of the major causes for the progressive cognitive decline in AD patients. The development of compounds interfering with A\u03b2 aggregation and thus able to rescue neurodegeneration is indispensible. For this purpose, fast and reliable assays are needed capable of showing direct effects of compounds on A\u03b2 oligomer formation.<\/p>\n\n\n\n<p>QPS Neuropharmacology provides exclusively a fast and reproducible screening assay,&nbsp;<a href=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/services\/ex-vivo-services\/molecular-biology-biomarker-analytics\/amorfix-aggregated-a%ce%b2-assay-a4-assay\/\" target=\"_blank\" rel=\"noreferrer noopener\">Amorfix Aggregated A\u03b2 Assay (A4)<\/a>, able to directly visualize beneficial effects of your developmental compounds on the formation of A\u03b2 aggregates&nbsp;<em>in vitro<\/em>. Notably, compound Y and Z were able to reduce A\u03b2 aggregate formation as determined by A4 assay.<\/p>\n\n\n\n<figure class=\"gb-block-image gb-block-image-5435e870\"><img loading=\"lazy\" decoding=\"async\" width=\"417\" height=\"347\" class=\"gb-image gb-image-5435e870\" src=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/A4-assay_in-vitro.png\" alt=\"A4-assay_in-vitro\" title=\"A4-assay_in-vitro\" srcset=\"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/A4-assay_in-vitro.png 417w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/A4-assay_in-vitro-300x250.png 300w\" sizes=\"(max-width: 417px) 100vw, 417px\" \/><\/figure>\n\n\n\n<p><strong>Figure: Determination of A\u03b2 oligomers&nbsp;<em>in vitro<\/em>&nbsp;by A4 Assay.<\/strong>&nbsp;Compounds were co-aggregated with A\u03b21-42 for 48h&nbsp;<em>in vitro<\/em>. Aggregated A\u03b2 was separated from monomers through affinity interaction. After disaggregation, the originally aggregated A\u03b2 was detected using a bead-based immunoassay that applies human A\u03b2-specific antibody labeled beads and Time Resolved Fluorescence (TRF) measurements. A\u03b2 levels were evaluated as signal to noise ratio (S\/N). Co-aggregation of compound Y and Z as well as reference item (RI) with A\u03b21-42 reduced aggregate formation.<\/p>\n\n<\/div><\/div>\n<\/div>\n\n<div class=\"gb-container gb-container-ccf7a260 gb-accordion__item\" data-transition=\"slide\">\n\n<button class=\"gb-button gb-button-15252a7f gb-accordion__toggle\"><span class=\"gb-button-text\"><strong>tau \uacfc\uc778\uc0b0\ud654<\/strong><\/span><span class=\"gb-icon\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon\"><path d=\"M416 208H272V64c0-17.67-14.33-32-32-32h-32c-17.67 0-32 14.33-32 32v144H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h144v144c0 17.67 14.33 32 32 32h32c17.67 0 32-14.33 32-32V304h144c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon-open\"><path d=\"M416 208H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h384c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><\/span><\/button>\n\n\n<div class=\"gb-accordion__content\"><div class=\"gb-container gb-container-9e1e834a\">\n\n<p>Tau proteins belong to the family of microtubule-associated proteins and play an important role in stabilizing the neuronal microtubules network. They are the major constituents of intraneuronal and glial fibrillar lesions described in Alzheimer\u2019s disease and numerous neurodegenerative disorders referred to as \u2018tauopathies\u2019, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick\u2019s disease (PiD), argyrophilic grain disease, as well as the inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).<\/p>\n\n\n\n<p>Molecular analysis revealed that hyperphosphorylation might be the important event leading to Tau aggregation resulting in neurodegeneration and dementia. Development of new compounds capable of preventing Tau hyperphosphorylation is an increasingly hot topic.<\/p>\n\n\n\n<p>Thus, reliable models are needed that reflect Tau hyperphosphorylation in human diseases. For this purpose, we generated the stably transfected SH-SY5Y cell line over-expressing the longest human Tau441 isoform comprising two disease related mutations (V337M\/R406W). The phosphorylation pattern of Tau in SH-SY5Y-TMHT441 cells can be reliably modulated by several kinase inhibitors. Effects on Tau phosphorylation (e.g. at Thr181, Ser202, Thr231\/Ser235, Ser262 and Ser396\/Ser4049) can be determined by various methods including immunosorbent assay (MSD), immunoblot analysis or mass spectrometry (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC3323815\/\" rel=\"noreferrer noopener\" target=\"_blank\">L\u00f6ffler T. et al, J Mol Neurosci 2012 May;47(1):192-203<\/a>).<\/p>\n\n\n\n<figure class=\"gb-block-image gb-block-image-decc4a44\"><img loading=\"lazy\" decoding=\"async\" width=\"685\" height=\"400\" class=\"gb-image gb-image-decc4a44\" src=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/SHSY-5Y-TMHT441.png\" alt=\"SHSY-5Y-TMHT441\" title=\"SHSY-5Y-TMHT441\" srcset=\"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/SHSY-5Y-TMHT441.png 685w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/SHSY-5Y-TMHT441-300x175.png 300w\" sizes=\"(max-width: 685px) 100vw, 685px\" \/><\/figure>\n\n\n\n<p><strong>Figure: Tau hyperphosphorylation in SH-SY5Y-TMHT441 cells.<\/strong>&nbsp;Immunocytochemistry showing overexpression of human Tau in (A) untransfected and (B) transfected SH-SY5Y cells (Scale bar 100 \u00b5m). Human Tau is indicated in red, DAPI in blue. (C) Tau phosphorylation in control SH-SY5Y cells, in undifferentiated and differentiated SH-SY5Y-CMV-TMHT441 cells was examined by immuno blot analysis. (D) pThr231 was reduced in SH-SY5Y-CMV-TMHT441 cells after treatment with the JNK inhibitor SP600125 as assessed by an immunosorbent assay (MSD).<\/p>\n\n<\/div><\/div>\n<\/div>\n\n<div class=\"gb-container gb-container-98713531 gb-accordion__item\" data-transition=\"slide\">\n\n<button class=\"gb-button gb-button-da453b4c gb-accordion__toggle\"><span class=\"gb-button-text\"><strong>\uc800\uccb4\uc628\uc99d<\/strong><\/span><span class=\"gb-icon\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon\"><path d=\"M416 208H272V64c0-17.67-14.33-32-32-32h-32c-17.67 0-32 14.33-32 32v144H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h144v144c0 17.67 14.33 32 32 32h32c17.67 0 32-14.33 32-32V304h144c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon-open\"><path d=\"M416 208H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h384c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><\/span><\/button>\n\n\n<div class=\"gb-accordion__content\"><div class=\"gb-container gb-container-e2c98f2c\">\n\n<p>Hyperphosphorylation and accumulation of Tau in neurons is one of the main pathologic hallmarks in Alzheimer\u2019s disease (AD) and other tauopathies, including Pick\u2019s disease (PiD), argyrophilic grain disease, familial frontotemporal dementia and parkinsonism linked to chromosome 17.<\/p>\n\n\n\n<p>Hyperphosphorylated Tau dissociates from microtubuli, resulting in the breakdown of the axonal flow, and thus impairs neuronal viability and function. Abnormal tau hyperphosphorylation is mainly induced due to the imbalance between protein kinases and phosphatases. To find promising drug candidates, inducible cellular models of Tau hyper-phosphorylation are useful screening tools for studying central nervous system drug effects.<\/p>\n\n\n\n<p>Tau hyper-phosphorylation was induced by hypothermic conditions (30\u00b0C) in SH-SY5Y cells or SH cells overexpressing the longest isoform of human Tau 441 carrying two well-characterized mutations V337M\/R406W (SH-SY5Y-Tau441). To reverse Tau hyperphosphorylation cells were treated with different kinase inhibitors like LiCl, a well known GSK3 beta inhibitor and were either kept under normo- or hypothermic conditions. Afterwards, total Tau and its phosphorylated species pSer262, pSer202, pSer396 and pThr231 were analyzed in cellular lysates by immunoblot or immunosorbent assay (Meso Scale Discovery, MSD).<\/p>\n\n\n\n<figure class=\"gb-block-image gb-block-image-55af0e9a\"><img loading=\"lazy\" decoding=\"async\" width=\"1009\" height=\"465\" class=\"gb-image gb-image-55af0e9a\" src=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Hypothermia-in-vitro.png\" alt=\"Hypothermia-in-vitro\" title=\"Hypothermia-in-vitro\" srcset=\"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Hypothermia-in-vitro.png 1009w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Hypothermia-in-vitro-300x138.png 300w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Hypothermia-in-vitro-768x354.png 768w\" sizes=\"(max-width: 1009px) 100vw, 1009px\" \/><\/figure>\n\n\n\n<p><strong>Figure: Hypothermia induced Tau hyper-phosphorylation is significantly reduced by LiCl in SH-SY5Y-Tau441 (top) and SH-SY5Y cells (bottom).<\/strong>&nbsp;Cells were subjected to 2h of hypothermia (30\u00b0C) and treated with either LiCl or Vehicle Control. Quantification of immunosorbent assay is shown of total Tau, pTau202, pTau181 and pTau396. Data are normalized to normothermic conditions. Data are shown as mean + SEM (n=4). Statistical significance is indicated by *&lt;0.05, **&lt;0.01, ***&lt;0.001 as determined by One-Way ANOVA (Newman\u2019s Keuls Multiple Comparison Test).<\/p>\n\n<\/div><\/div>\n<\/div>\n\n<div class=\"gb-container gb-container-14c140fd gb-accordion__item\" data-transition=\"slide\">\n\n<button class=\"gb-button gb-button-96f96538 gb-accordion__toggle\"><span class=\"gb-button-text\"><strong>\uccb4\uc678 tau \uc751\uc9d1<\/strong><\/span><span class=\"gb-icon\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon\"><path d=\"M416 208H272V64c0-17.67-14.33-32-32-32h-32c-17.67 0-32 14.33-32 32v144H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h144v144c0 17.67 14.33 32 32 32h32c17.67 0 32-14.33 32-32V304h144c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 448 512\" width=\"1em\" height=\"1em\" ariahidden=\"true\" role=\"img\" class=\"gb-accordion__icon-open\"><path d=\"M416 208H32c-17.67 0-32 14.33-32 32v32c0 17.67 14.33 32 32 32h384c17.67 0 32-14.33 32-32v-32c0-17.67-14.33-32-32-32z\" fill=\"currentColor\"><\/path><\/svg><\/span><\/button>\n\n\n<div class=\"gb-accordion__content\"><div class=\"gb-container gb-container-2ad1d13e\">\n\n<p>Tau protein stabilizes microtubules and contributes to key structural and regulatory cellular functions as axonal transport and signaling. Neurofibrillary tangles, mainly composed of bundles of Tau are implicated in the pathogenesis of neurodegenerative diseases such as tauopathies including Alzheimer\u2018s disease.<\/p>\n\n\n\n<h5 class=\"gb-headline gb-headline-324957c1 gb-headline-text\">To identify inhibitors of Tau aggregation QPS provides two approaches:<\/h5>\n\n\n\n<p>The first approach is a spectrofluorometric read out that can be performed as Tau aggregation or dis-aggregation assay. The Tau aggregation assay aims to identify compounds that are able to mitigate the aggregation of Tau. The Tau dis-aggregation assay investigates if developmental compounds are able to reverse Tau aggregation.<\/p>\n\n\n\n<p>These assays are cell-free&nbsp;<em>in vitro<\/em>&nbsp;assays using recombinant Tau441 (2N4R) P301L that is incubated in an aggregation buffer including ThioS. Fluorescence intensity is detected at 465 nm excitation and 510 nm emission.<\/p>\n\n\n\n<figure class=\"gb-block-image gb-block-image-f1a37cb7\"><img loading=\"lazy\" decoding=\"async\" width=\"757\" height=\"339\" class=\"gb-image gb-image-f1a37cb7\" src=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Spectro.png\" alt=\"Spectro\" title=\"Spectro\" srcset=\"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Spectro.png 757w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/Spectro-300x134.png 300w\" sizes=\"(max-width: 757px) 100vw, 757px\" \/><\/figure>\n\n\n\n<p><strong>Figure 1: Test Item 2 minimized Tau aggregation&nbsp;<em>in vitro<\/em>&nbsp;and was also able to reduce pre-aggregated Tau.<\/strong>&nbsp;Test item 1 had no effect on Tau aggregation. EC50 was determined showing effective concentrations at 1 and 0.65 \u00b5M in the Tau aggregation and Tau dis-aggregation assay, respectively.<\/p>\n\n\n\n<p>In a second approach fibrillization of Tau was monitored using transmission electron microscopy (TEM). Samples were placed onto carbon \u2013 coated grids and a negative staining was performed (1% Uac). The TEM analysis was done in cooperation with the Institute of Cell Biology, Histology &amp; Embryology at the Medical University of Graz.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<figure class=\"gb-block-image gb-block-image-a561fbd5\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"715\" class=\"gb-image gb-image-a561fbd5\" src=\"http:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/ELMI-1024x715-1.png\" alt=\"ELMI\" title=\"ELMI-1024x715\" srcset=\"https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/ELMI-1024x715-1.png 1024w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/ELMI-1024x715-1-300x209.png 300w, https:\/\/scantoxneuro.vm0857.enterprisecloud.nu\/wp-content\/uploads\/2020\/02\/ELMI-1024x715-1-768x536.png 768w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/figure>\n\n\n\n<p><strong>Figure 2: Test Item 2 reduced the size and number of Tau fibrils in the Tau aggregation (upper row) and dis-aggregation assay (lower row) in contrast to the Vehicle Control.<\/strong>&nbsp;Long Tau fibrils in the Vehicle Control are indicated by blue triangles and short ones after applying test Item 2 are indicated by orange triangles.<\/p>\n\n<\/div><\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div><\/div>\n\n<div class=\"gb-grid-column gb-grid-column-be7dde5f\"><div class=\"gb-container gb-container-be7dde5f\">\n<div class=\"gb-container gb-container-5783dd95 Sidebar In Vitro\">\n<div class=\"gb-container gb-container-2ff2e5d1\">\n\n<h3 class=\"gb-headline gb-headline-240379c5 gb-headline-text gb-headline-98ccc1d8 blueline\">Newsletter<\/h3>\n\n\n\n<div class=\"gb-grid-wrapper gb-grid-wrapper-65d3a036 gb-query-loop-wrapper\">\n<div class=\"gb-grid-column gb-grid-column-631b4a4b gb-query-loop-item post-8004 qps_newsletter type-qps_newsletter status-publish hentry\"><div class=\"gb-container gb-container-631b4a4b\">\n<div 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