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BioAssay: AID 2713

qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytotoxicity in HTT Striatal Cells

Huntington's disease is a neurodegenerative disorder caused by a trinucleotide repeat expansion in Exon 1 of the Huntingtin gene. The CAG trinucleotide encodes glutamine and polyglutamine expansions cause cell death in selective areas of the brain. Huntington polyglutamine repeats have the tendency to form aggregates which are observable in later stages of the disease. The exact nature of the aggregates, whether toxic, protective, or insignificant, is unclear. ..more
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 Tested Compounds
 Tested Compounds
All(4)
 
 
Active(1)
 
 
Inconclusive(3)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Active(2)
 
 
Inconclusive(3)
 
 
AID: 2713
Data Source: NCGC (HTT014)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-27

Data Table ( Complete ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeProbeComment
1482Quantitative High-Throughput Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Summarysummary2
1471qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection (ATP)confirmatory
1688qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Aggregate Formation (GFP)confirmatory
2673qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection confirmation (ATP)confirmatory
2669qHTS Assay Multiplex Screening to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection (Protease relase)confirmatory
2672qHTS Assay Multiplex Screening to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection in HTT Q25 expressing cells (ATP)confirmatory
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: MH084839-01A1
Assay Submitter (PI): Wei Zheng

NCGC Assay Overview:

Huntington's disease is a neurodegenerative disorder caused by a trinucleotide repeat expansion in Exon 1 of the Huntingtin gene. The CAG trinucleotide encodes glutamine and polyglutamine expansions cause cell death in selective areas of the brain. Huntington polyglutamine repeats have the tendency to form aggregates which are observable in later stages of the disease. The exact nature of the aggregates, whether toxic, protective, or insignificant, is unclear.

A stable PC12 cell line containing a gene fusion of Exon 1 of the Huntingtin gene linked to GFP under the control of the inducible ecdysone promoter were used as the cell-based model of Huntington Disease for high throughput screening. Exon 1 of the Huntingtin gene contained an expansion of 103 polyglutamines (Q103) which, when expressed, caused cell death and distinct, bright GFP aggregates. The amount of cell death and the size and intensity of GFP aggregates increased over time and induction level. The cell death were quantified with a measurement of ATP content in the cells. A maximal 40-50% of cell death was observed when the Huntington gene was induced in this cell line.

We optimized this assay in a homogeneous 1536 well format. A quantitative high throughput screen (qHTS) was conducted on the PC12 cells which were induced to express the toxic fusion construct. Small molecules which prevented cell death were identified by using a luminescent ATP quantification method (Perkin Elmer's ATPlite).

This assay examines influence on cytotoxicity induced by serum deprivation in normal and Huntington striatal cells.
Protocol
Freshlay passaged STHdhQ7/7 cells and mutant STHdhQ111/111 Huntington striatal cells were plated at a density of 20,000 cells/well in complete media and allowed to recover overnight at 33C. The next day, media was replaced with serum containing media or serum free media containing the compound. After 24 hours of incubation at 33 C, cells were assayed for ATP content using the Cell Titer-GLO ATP assay from Promega (cat # G7571).

ATP/Toxicity Assay in Serum Free Media (SFM)
(96 well plate format)
1. Plate 20,000 cells/well and incubate the cells in complete (w/serum) media overnight
2. Remove media, wash twice with PBS and add the SFM w/ or w/o the compound
3. Incubate at 33C; cells can be incubated at 39C for a more dramatic effect
4. ATP measurements after 4h treatment, per kit protocol
Comment
Active compounds which improve the viability of STHdhQ111/111 cells with little effect on STHdhQ7/7 cells are given a score of 80. Compounds that enhance cytotoxicity are given a score of 10. Inactive compounds are given a score of 0.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeType of activity observed, one of: cytoprotective, cytotoxic, inactive.String
2Activity at 30uM in STHdhQ7/7 cultured in FBS (30μM**)Percent change in cellular viability at tested concentration for cells cultured in fetal bovine serum expressing non-cytotoxic HTT proteinFloat%
3Activity at 30uM in STHdhQ111/111 cultured in FBS (30μM**)Percent change in cellular viability at tested concentration for cells cultured in fetal bovine serum expressing cytotoxic HTT proteinFloat%
4Activity at 30uM in STHdhQ7/7 cultured in SFM (30μM**)Percent change in cellular viability at tested concentration for cells cultured in serum free medium expressing non-cytotoxic HTT proteinFloat%
5Activity at 30uM in STHdhQ111/111 cultured in SFM (30μM**)Percent change in cellular viability at tested concentration for cells cultured in serum free medium expressing cytotoxic HTT proteinFloat%
6Activity at 60uM in STHdhQ7/7 cultured in FBS (60μM**)Percent change in cellular viability at tested concentration for cells cultured in fetal bovine serum expressing non-cytotoxic HTT proteinFloat%
7Activity at 60uM in STHdhQ111/111 cultured in FBS (60μM**)Percent change in cellular viability at tested concentration for cells cultured in fetal bovine serum expressing cytotoxic HTT proteinFloat%
8Activity at 60uM in STHdhQ7/7 cultured in SFM (60μM**)Percent change in cellular viability at tested concentration for cells cultured in serum free medium expressing non-cytotoxic HTT proteinFloat%
9Activity at 60uM in STHdhQ111/111 cultured in SFM (60μM**)Percent change in cellular viability at tested concentration for cells cultured in serum free medium expressing cytotoxic HTT proteinFloat%

** Test Concentration.
Additional Information
Grant Number: MH084839-01A1

Data Table (Concise)
Classification
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