The novel hydroxylamine derivative NG-094 suppresses polyglutamine protein toxicity in Caenorhabditis elegans - PubMed (original) (raw)
The novel hydroxylamine derivative NG-094 suppresses polyglutamine protein toxicity in Caenorhabditis elegans
Pierre Haldimann et al. J Biol Chem. 2011.
Abstract
Aggregation-prone polyglutamine (polyQ) expansion proteins cause several neurodegenerative disorders, including Huntington disease. The pharmacological activation of cellular stress responses could be a new strategy to combat protein conformational diseases. Hydroxylamine derivatives act as co-inducers of heat-shock proteins (HSPs) and can enhance HSP expression in diseased cells, without significant adverse effects. Here, we used Caenorhabditis elegans expressing polyQ expansions with 35 glutamines fused to the yellow fluorescent protein (Q35-YFP) in body wall muscle cells as a model system to investigate the effects of treatment with a novel hydroxylamine derivative, NG-094, on the progression of polyQ diseases. NG-094 significantly ameliorated polyQ-mediated animal paralysis, reduced the number of Q35-YFP aggregates and delayed polyQ-dependent acceleration of aging. Micromolar concentrations of NG-094 in animal tissues with only marginal effects on the nematode fitness sufficed to confer protection against polyQ proteotoxicity, even when the drug was administered after disease onset. NG-094 did not reduce insulin/insulin-like growth factor 1-like signaling, but conferred cytoprotection by a mechanism involving the heat-shock transcription factor HSF-1 that potentiated the expression of stress-inducible HSPs. NG-094 is thus a promising candidate for tests on mammalian models of polyQ and other protein conformational diseases.
Figures
FIGURE 1.
Temperature of culture affects polyQ protein aggregation and toxicity. A, effect of culture temperature on the motility of 3-day-old C. elegans animals expressing polyQ-YFP fusion proteins with 0 (Q0) or 35 glutamine residues (Q35) in body wall muscle cells. B, effect of culture temperature on age-dependent changes in the motility of Q0 and Q35 animals. Values in A and B are means ± S.E. (error bars) of 12 motility assays from two independent experiments, two replicates/experiment, and three assays/experiment. Note that cohort sizes decreased as some animals died during the experiment. This was mainly observed for Q35 animals grown at elevated temperature. Dead animals were excluded from the analysis. C, fluorescence microscopy images of Q0 and Q35 animals (2 days post-hatch) cultured at 20 °C or 25 °C. The white arrow indicates the region of the head also shown at higher magnification. Note that the head of the Q35 animal cultured at 25 °C is slightly rotated. Images were taken using the 10× objective of a Leica DM5000B microscope. D, fluorescence microscopy images of embryos laid by Q0 and Q35 animals cultured at 20 °C or 25 °C. Images were taken using the 20× objective of a Leica DM5000B microscope.
FIGURE 2.
NG-094 alleviates polyQ-dependent animal paralysis. A, chemical structure of different hydroxylamine derivatives. B, motility index of 3-day-old Q0 and Q35 animals cultured at 22 °C and treated with different concentrations of NG-094. C, motility index of 3-day-old Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094, as indicated. Parent animals were cultured at 20 °C. D, motility index of 6-day-old Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094, as indicated. Values are means ± S.E. (error bars) of nine motility assays from three independent experiments and three assays per experiment. ***, p < 0.001; ns, not significant.
FIGURE 3.
NG-094 confers cytoprotection without significant adverse effects on the fitness of the nematodes. A, images of 3-day-old Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094. Arrows indicate animals with a delayed development, and arrowheads indicate embryos. B, reproductive profile of Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094. Values are means ± S.E. (error bars) of at least 15 individuals. C, brood size per animal for Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094. Values are means ± S.E. of at least 15 individuals. *, p < 0.05; ***, p < 0.001; ns, not significant. D, survival of Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094. Statistical data are given in
supplemental Table S1
. E, fluorescence microscopy images of the head of 3-day-old Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094. Images were taken using the 10× objective of a Leica DM500B microscope. F, number of Q35-YFP aggregates exhibited by Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094. Values are means ± S.E. of 50 animals. ***, p < 0.001.
FIGURE 4.
Heat- and NG-094-induced changes in the transcription of HSP genes. Relative mRNA levels of HSP70(C12C8.1) (A), HSP70(F44E5.4) (B), HSP16.2 (C), HSP16.11 (D), HSP40(F39B2.10.1) (E), and HSP90(daf-21) (F) measured by RT-qPCR for 3-day-old Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094. Analyses were performed for animals that on the 3rd day since hatching were either kept at 22 °C or heat-shocked at 29 °C or 35 °C for 90 min. Data were normalized to Act-4 levels. Values were calculated relative to the value of untreated Q0 animals. Similar results were obtained when data were normalized to 18 S mRNA levels (data not shown). Data are means ± S.E. (error bars) of four independent biological samples.
FIGURE 5.
NG-094 enhances heat-induced accumulation of HSPs. A, Western blot analysis of the levels of HSPs in 3-day-old Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094. Analyses were performed for animals that on the 3rd day since hatching were either kept at 22 °C or heat-shocked at 35 °C for 2 h or at 29 °C for 3 h. Animals were collected after a 6-h period of recovery at 22 °C. B, expression levels of HSP40 and HSP16.2 in Q0 and Q35 animals cultured at different temperatures from embryo until early adulthood. C, expression levels of YFP and Q35-YFP in 3-day-old Q0 and Q35 animals cultured at 22 °C and treated or not with 1 m
m
NG-094. D, expression levels of GFP in 3-day-old BC10060 nematodes expressing a C12C8.1(hsp70)_::_GFP transgene. Animals were cultured at 22 °C and treated or not with 1 m
m
NG-094. Analyses were performed for animals that on the 3rd day since hatching were either kept at 22 °C or heat-shocked at 29 °C for 3 h. Animals were collected after a 6-h period of recovery at 22 °C. α-Tubulin was used as a loading control.
FIGURE 6.
Cytoprotection by NG-094 requires HSF-1 but not DAF-16 activity. A, motility index of 6-day-old Q35 animals that were grown at 20 °C on either control bacteria (EV) or hsf-1 RNAi bacteria and treated or not with 1 m
m
NG-094. NG-094 was administered starting from the 3rd day since hatching. Values are means ± S.E. (error bars) of nine motility assays from three independent experiments and three assays per experiment. ***, p < 0.001; ns, not significant. B, Western blot analysis of the expression level of HSP16.2 in 6-day-old Q35 animals that were grown on control bacteria (EV) or hsf-1 RNAi bacteria. Analyses were performed for animals that on the 6th day since hatching were either kept 20 °C or heat-shocked at 35 °C for 2 h. Animals were collected after a 6-h period of recovery at 22 °C. α-Tubulin was used as a loading control. C, images of 3-day-old TJ356 nematodes expressing a _daf-16::_GFP transgene. Animals were grown at 22 °C on control bacteria (EV) or daf-16 RNAi bacteria. Shown are fluorescence images (upper panels) and overlays of fluorescence and diffraction contrast images (lower panels). All pictures were taken with the same exposure. D, motility index of 3-day-old Q35 animals that were grown at 22 °C on control bacteria (EV) or daf-16 RNAi bacteria and treated or not with 1 m
m
NG-094. Values are means ± S.E. of nine motility assays from three independent experiments and three assays per experiment. ***, p < 0.001.
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