Screen for compounds that correct neuronal dysfunction without cell death as induced by the expression of polyglutamine-expanded, N-terminal huntingtin in C. elegans mechanosensory neurons (HDELENEU)
Huntington's disease (HD) is a dominant neurodegenerative disease that results from the presence of a CAG expansion, encoding expanded polyglutamines (polyQ), in the first exon of the huntingtin gene. HD results in selective neuronal loss within the striatum and various cortical areas. HD pathogenesis appears to be initiated by the formation of N-terminal fragments of polyQ-expanded huntingtin. more ..
Huntington's disease (HD) is a dominant neurodegenerative disease that results from the presence of a CAG expansion, encoding expanded polyglutamines (polyQ), in the first exon of the huntingtin gene. HD results in selective neuronal loss within the striatum and various cortical areas. HD pathogenesis appears to be initiated by the formation of N-terminal fragments of polyQ-expanded huntingtin. Before cell death, cell dysfunction could significantly contribute to the early stages of the disease as observed in several mouse HD models. Morphologic abnormalities have been demonstrated including dystrophic neurites in human HD brain sections, decrease of dendrites in mouse HD models, and inhibition of neuritic growth in cellular HD models. Like cell death, cell dysfunction may result from the toxicity of cellular aggregates, soluble mutant huntingtin, or both. A C. elegans HD model generated in our laboratory indicates that significant neuronal dysfunction without cell death is a phenotype that can be induced by the expression of polyQ-expanded N-terminal huntingtin (CFP fusions) in mechanosensory neurons. C. elegans transgenics displayed morphologic abnormalities of axons, and neuritic aggregates. In this model, the read-out for neuronal dysfunction was the unability of C. elegans transgenics to respond by backing away from gentle touch at the tail, a defective phenotype modulated by the length of the polyQ tract. We developed a screen for compounds that correct this behavioural defect in C. elegans.
Screening - incubation of C. elegans transgenics with test compounds. C. elegans transgenics (128 consecutive Glns) were synchronized at the L1 larvae stage. L1s were manually seeded in 96-well assay plates (Costar white plates, #3596) 50 microliters/well for an animal density of around 20 L1s/well. Compounds from the NINDS library were added to reach a final concentration of 100-12.5 micro-M depending on solubility (diluted with assay medium that contains no bacteria and no DMSO, by 100-800x from the 10 mM stock solution in DMSO). Animals were incubated with the test compounds at 20 degrees C for ~72 hours (4 wells/compound). After incubation, animals (now young adults) from 2 wells were transfered to a single Petri dish without bacteria (2 dishes/compound)and allowed to rest for about 15'. Animals were then tested for response to gentle touch at the tail using a fine eyebrow hair mounted on a tooth pick (2 intra-assay readings/compound), and the percentage of animals backing away from touch at the tail was determined.
Growth Medium: The growth medium for C. elegans nematodes was S medium (S basal supplemented with potassium citrate 10 micro-M, CaCl2 3.3 micro-M, MgSO4 3.3 micro-M; pH 6) with bacteria (strain OP50-1), streptomycin 111 units/ml (Sigma, cat. no. S6501), nystatin 166 units/ml (Sigma, cat. no. N3503), and 1.25% DMSO (Sigma, cat. no. D5879).
Data Analysis: The baseline in the assay ('negative control') was determined from incubation of trangenic animals expressing 128 Glns with no compound in the growth medium (10 readings; >50 animals/reading; the highest and lowest of the ten readings were dropped), and expressed as the percentage of animals backing away from touch at the tail (percentage of responders). The baseline was found to be 18 +/- 4.2%. The positive control was determined from incubation of transgenic animals expressing 19 Glns with no compounds in the growth medium (10 readings; >50 animals/reading; the highest and lowest of the ten readings were dropped), and was found to be 59 +/- 5.7%. The entire primary assay was perfomed once and each compound library plate was tested in duplicate, yealding two readings that were averaged. These averaged values of compound-treated response versus baseline response were denoted R. The difference of R and baseline measures the change in response upon compound treatment and was noted S, the final score that is output. As expected, three categories of compounds were identified: compounds found to be toxic at the concentration tested (R and S values set to zero), compounds that worsened the mechanosensory defective phenotype induced by 128 Glns (percentage of responders lower than 13.8%; negative S value), and compounds that corrected the mechanosensory defective phenotype induced by 128 Glns (percentage of responders greater than 22.2%; positive S value).
Dilution Series: All compounds showing a positive score S greater than the baseline+SD value (22.2%) in the primary screen were selected for retesting in a 2-fold, 4-5 point dilution series with a maximum concentration of 100 to 12.5 micro-M. Once created, the assay was performed as described in the screening section. Compounds showing no activity (false positives) and compounds showing poorly-informative dose-response curves (for example, compounds active only at the highest concentration tested) were dropped. The assay was performed again for compounds showing informative (sigmoid type) or apparently incomplete (activity occurring at low doses) dose-response curves, by extending the range of dilution series when necessary. The percentages of responders at each of the concentrations tested were averaged, and concentrations required to achieve 50% of the maximally achievable effect (EC50) for each compound were determined. This selection scheme resulted in the identification of 27 compounds showing significant activities and reproducible dose-response curves with EC50s ranging from 0.005 to 40 microM, and maximally achievable effects greater than 30%.
Number Replicates: 1
Assay Readout: Forward movement induced by gentle touch at the tail
Concentration Primary Screen: (uM) 100
Number Negative Controls: 8
Negative Control Mean: 18.0
Negative Control Standard Deviation: 4.2
Number Positive Controls: 8
Positive Control Mean: 59.0
Positive Control Standard Deviation: 5.7
Concentration 10O-12.5 depending on compound solubility in the growth medium to rank compounds, the EC50 and the maximally effect achievable may be used as equally important criteria. Compound activities are being characterized further by looking at agregation of CFP fusions, morphologic abnormalities of mechanosensory neurons at the tail in transgenic animals expressing 128 Glns, and behavioural response of C. elegans transgenics that express CFP fusions with 19Glns or that express no CFP fusions. NEMATODE STRAIN: Transgenic C. elegans strain (integrated array) derived from C. elegans lin-15 (n765) hermaphrodites (C. elegans Genetic Center, St. Paul, MN) injected with a wild type lin-15 marker, a mec-7 promoter_YFP plasmid, and a mec-3 promoter_Huntingtin 1-57(128 Glns)::CFP plasmid.
A Note about the Activity Matrix Rankings:
The data for each assay has been converted into a numerical ranking of all 1040 compounds for that assay. Thus, a compound with a rank of 1 was the top scoring compound in that assay and a compound with a rank of 1040 was the lowest scoring compound in that assay. Compounds with equal scoring were given equal rank.
PubChem scores were derived as (1040-NINDS activity rank). Substances with reported EC50 values were considered active.
PI Name Christian Neri
Affiliation Human Polymorphim Study Center (CEPH)
Investigator Sandra Boiziau
Investigator Emmanuel Lambert
Investigator Alex Parker
Data Table (Concise)