Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models

Figures & data

Figure 1 Indole and derivative NC001-8 and cytotoxicity.

Notes: (A) Structure, formula, and molecular weight of indole and synthetic derivative NC001-8. (B) Cytotoxicity of GGA, indole, and NC001-8 against SH-SY5Y cells, using Hoechst-propidium iodide staining. Cells were treated with 100 nM~100 μM tested compounds, and cell proliferation was measured the next day (n=3). The half maximal inhibitory concentration of each compound was shown under the columns. To normalize, the relative viability in untreated cells is set as 100%.
Abbreviations: GGA, geranylgeranylacetone; IC₅₀, inhibitory concentration at 50% level.

Figure 2 Enhancement of chaperone expression by indole and NC001-8 in HEK-293 cells.

Notes: (A) Fluorescent reporters mCherry, ZsYellow1, and AmCyan1 driven by HSF1, HSPA8, and HSPA1A promoter fragments, respectively (top), and effects of GGA, indole, and NC001-8 (100 nM~100 μM) on HSF1, HSPA8, and HSPA1A promoter activities (bottom). To normalize, the fluorescence level in untreated cells is set as 100%. Three independent experiments were performed, with P<0.05 considered significant. (B) Representative Western blot images of HEK-293 cells treated with GGA, indole and NC001-8 (100 nM) for two days, using HSF1, HSPA8, HSPA1A, and β-actin antibodies. Levels of HSF1, HSPA8, and HSPA1A were normalized with a loading control (β-actin). Data are expressed as the mean ± standard deviation values from three independent experiments.
Abbreviations: GGA, geranylgeranylacetone; 001-8, NC001-8; HSF1, heat shock transcription factor 1; Rel., relative; HSPA8, heat shock 70 kDa protein 8; HSPA1A, heat shock 70 kDa protein 1A.

Figure 3 SH-SY5Y cells with induced TBP/Q_36~79-GFP expression and neuronal phenotype.

Notes: (A) Real-time polymerase chain reaction quantification (n=3) of TBP/Q_36~79-GFP mRNA level relative to HPRT1 mRNA after 2 days of induction with doxycycline (+ Dox) or not (− Dox). (B) Western blot analysis of TBP/Q_36~79-GFP protein level using TBP (N-12) antibody after 2 days of induction with doxycycline (+ Dox) or not (− Dox). (C) Representative microscopic images (top) of TBP/Q_36~79-GFP cells after induced differentiation with retinoic acid (+ RA) for 7 days and aggregate quantification (bottom; n=3) of cells with induced differentiation for 7~21 days. P-values were evaluated by one-way analysis of variance with post hoc LSD test. (D) Representative microscopic images (top) of neuronal differentiated TBP/Q₃₆ and TBP/Q₇₉ cells (for 14 days) and quantification (n=3) of neuronal processes and branches (bottom) of cells with induced differentiation for 7~21 days (blue, nuclei; green, expressed TBP/Q_36~79-GFP protein; red, cell body and outgrowth segmentation).
Abbreviations: Dox, doxycycline; TBP, TATA box binding protein; GFP, green fluorescent protein; mRNA, messenger RNA; Ab, antibody; LSD, Fisher’s least significant difference.

Figure 4 Enhancement of HSF1 and chaperone expression and reduction of aggregation by indole and NC001-8 in neuronal SH-SY5Y TBP/Q₇₉ cells.

Notes: (A) Cells were pretreated with GGA, indole, or NC001-8 (100 nM) for 8 hours and TBP/Q₇₉-GFP expression induced for 6 days. Relative HSF1, HSPA8, and HSPA1A expressions were analyzed by immunoblot analysis, using β-actin as a loading control (n=3). P-values were evaluated by one-way analysis of variance with post hoc LSD test. (B) Cells were treated with GGA, indole, or NC001-8 (100 nM) for 7 days, and relative aggregation assessed by HCA system (n=3). To normalize, the relative aggregation level in untreated cells is set as 100%.
Abbreviations: Dox, doxycycline; GGA, geranylgeranylacetone; HSF1, heat shock transcription factor 1; HSPA8, heat shock 70 kDa protein 8; HSPA1A, heat shock 70 kDa protein 1A; Rel., relative; GFP, green fluorescent protein; HCA: high content analysis; 001-8, NC001-8; LSD, Fisher’s least significant difference.

Figure 5 Indole and NC001-8 promoted neurite outgrowth and reduced aggregation of Purkinje cells in spinocerebellar ataxia type 17 mouse cerebellar primary and slice cultures.

Notes: The primary culture was treated with 0~100 nM indole (A) or NC001-8 (B) for 13 days. The representative microscopic images of treatment with 100 nM indole or NC001-8 are shown, and the relative Purkinje cell neurite outgrowth (green) and aggregation (shown in white in the middle column and in red in the right merged column) were quantified (n=3). (C) The slice culture was treated with 10 nM indole or 10 μM NC001-8 for 6 days. The representative microscopic images of treatment are shown, and the relative Purkinje cell aggregation (red) was quantified (n=3). To normalize, the relative neurite outgrowth length and aggregation level in vehicle-treated cells or slices is set as 100%. IP3R-1 and 1TBP18 antibodies were used to detect the Purkinje cells and TBP aggregation, respectively.
Abbreviations: IP3R-1, inositol 1,4,5 trisphosphate receptor type 1; 1TBP18, mouse monoclonal antibody to TATA binding protein (TBP); Rel., relative; DAPI, 4′,6-diamidino-2-phenylindole.