Figure 1.
Concentration of compounds for testing was assessed using a food clearance assay.
(A) Flow diagram of the food clearance assay. 20 newly hatched L1 animals were incubated at 25°C in E. coli at a final OD (A595) of 0.6 in 96 well microtiter plate wells containing varying drug concentrations. The OD of the microtiter plate was measured daily for 5 days. (B) The OD of E. coli is reported daily for each concentration of LiCl. The mean OD is calculated for each day from triplicate samples and plotted over time. Error bars represent SEM. Food clearance assays were also performed on trichostatin A and mithramycin, (Figure S1). (C) Animals treated with no drug and indicated LiCl concentrations. Animals treated with 50 mM or 100 mM LiCl are alive but concentrations above 100 mM LiCl cause death (data not shown). Scale bar is approximately 75 µm.
Table 1.
Compounds screened in C. elegans polyQ model for neuroprotection.
Figure 2.
LiCl, TSA, and MTR decrease polyQ mediated ASH neuronal death.
(A) Flow diagram of the sensitized assay. 20–30 synchronized pqe-1;Htn-Q150 L1s per well were incubated at 15°C in S medium with E. coli at an OD (A595) of 0.6 and varying concentrations of compound to a total volume of 60 µl. Animals were grown in the presence of compound for 3 days. ASH neuron survival was evaluated by the presence or absence of GFP expression. (B–D) LiCl, TSA and MTR increased ASH neuron survival in the pqe-1;Htn-Q150 animals in the presence of E. coli. ASH neuron survival was evaluated by the presence or absence of GFP expression on day 3. 100 neurons were scored for each trial; mean percentage of 3 different trials±SEM is shown. p<0.0001 for 25 mM LiCl. p = .037 for 0.5 mM MTR; p<0.0001 for 1 mM MTR. p = 0.0082 for 165 µM TSA; p = 0.005 for 330 µM TSA.
Figure 3.
Compounds decrease polyQ toxicity in a pqe-1 independent manner in aged animals.
(A) Flow diagram of the aging assay. 30 synchronized pha-1;Htn-Q150 L1s per well were incubated at 25°C in S medium with E. coli at an OD (A595) of 0.5 with varying concentrations of compound to a total volume of 1 ml. On day 7, animals were collected and dye filled using DiD. (B) LiCl increased ASH neuron survival in the pha-1;Htn-Q150 aged animals. ASH neurodegeneration was evaluated by the presence or absence of dye-filled ASH neurons on day 7. 50 neurons were scored for each trial; mean percentage of 3 different trials±SD is shown. p = 0.0033 for 15 mM LiCl.
Figure 4.
LiCl and MTR decrease polyQ toxicity measuring ASH neuronal death in the absence of growth.
(A) Flow diagram of the starvation assay. 50 synchronized pqe-1;Htn-Q150 L1s per well were incubated at 25°C in S medium with varying concentrations of compound to a total volume of 60 µl. On day 2, the survival of the ASH neuron was determined by visually evaluating the presence or absence of GFP expression. (B) LiCl and MTR increase neuronal survival in starved conditions. 100 neurons were scored for each trial; mean percentage of 3 different trials±SEM is shown. p = 0.0063 for 10 mM LiCl; p<0.0001 for 25 mM, 50 mM and 100 mM LiCl. p<0.0001 for 0.5 mM, 0.75 mM, 1.0 mM, and 1.5 mM MTR. (C) LiCl and MTR increase neuronal survival in a daf-16 independent manner. At least 100 neurons were scored for each trial; percentage of neuronal survival in one representative experiment of 2 independent trials is shown; p<0.0001 for 25 mM and 50 mM LiCl. p<0.0001 for 0.5 mM and 1.0 mM MTR. (D) Combined, MTR (0.5 mM) and LiCl (25 mM) were more effective than either drug alone in protecting ASH neurons from polyQ neurotoxicity. 50 synchronized pqe-1;Htn-Q150 L1s per well were incubated at 25°C in S medium with 0.5 mM MTR and 25 mM LiCl to a total volume of 60 µl. On day 2, the survival of the ASH neuron was evaluated by the presence or absence of GFP. 100 neurons were scored for each trial; mean percentage of 3 different trials±SEM is shown. The excess synergy was 0.14 based on a Bliss Independence analysis. p = 0.046 for 0.5 mM MTR; p = 0.0039 for 25 mM LiCl; p<0.0001 for LiCl+MTR.
Figure 5.
Schematic Diagram of Compound Testing Strategy.
(1) A range of concentrations for each compound for testing in C. elegans was established using the food clearance assay. (2) The protective effects of compounds on polyglutamine neurodegeneration and cell death were assessed in pqe-1;Htn-Q150 animals treated with compounds for 3 days. (3) To determine if neuroprotective effects of compounds were dependent on pqe-1, compounds were retested in animals expressing Htn-Q150 for 7 days. (4) To distinguish drug effects on neuronal cell death versus effects on growth and/or development, synchronized pqe-1;Htn-Q150 L1 animals were incubated in the presence of drugs without food for 2 days. (5) The neuroprotective effects of compounds on the aging process can be tested by introducing mutations (daf-16) in components of specific aging-related (insulin signaling) pathways.