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

Kung PJ, Tao YC, Hsu HC, Chen WL, Lin TH, Janreddy D, Yao CF, Chang KH, Lin JY, Su MT, Wu CH, Lee-Chen GJ, Hsieh-Li HM - Drug Des Devel Ther (2014)

Bottom Line: The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events.We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells.Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.

ABSTRACT
In spinocerebellar ataxia type 17 (SCA17), the expansion of a translated CAG repeat in the TATA box binding protein (TBP) gene results in a long polyglutamine (polyQ) tract in the TBP protein, leading to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On SH-SY5Y cells with inducible SCA17 TBP/Q79-green fluorescent protein (GFP) expression to test indole and synthetic derivative NC001-8 for neuroprotection. We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells. The effects on promoting neurite outgrowth and on reduction of aggregation on Purkinje cells were also confirmed with cerebellar primary and slice cultures of SCA17 transgenic mice. Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment.

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SH-SY5Y cells with induced TBP/Q36~79-GFP expression and neuronal phenotype.Notes: (A) Real-time polymerase chain reaction quantification (n=3) of TBP/Q36~79-GFP mRNA level relative to HPRT1 mRNA after 2 days of induction with doxycycline (+ Dox) or not (− Dox). (B) Western blot analysis of TBP/Q36~79-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/Q36~79-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/Q36~79-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.
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f3-dddt-8-1929: SH-SY5Y cells with induced TBP/Q36~79-GFP expression and neuronal phenotype.Notes: (A) Real-time polymerase chain reaction quantification (n=3) of TBP/Q36~79-GFP mRNA level relative to HPRT1 mRNA after 2 days of induction with doxycycline (+ Dox) or not (− Dox). (B) Western blot analysis of TBP/Q36~79-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/Q36~79-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/Q36~79-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.

Mentions: To test the aggregation reduction potential of indole and NC001-8 in neuronal cells, we constructed Flp-In SH-SY5Y SCA17 cells with N-terminal TBP/Q36~79-GFP expression in an inducible fashion. As shown in Figure 3A, real-time polymerase quantification of these TBP lines shows 9~11 times TBP expression after induction with doxycycline for 2 days. In immunoblot, TBP antibody detected 52~62 kDa TBP/Q36~79-GFP protein in addition to the endogenous 43 kDa TBP protein (Figure 3B). When TBP/Q36~79 SH-SY5Y cells were differentiated for 7 to 21 days, using retinoic acid,39,40 a Q length-dependent and expression time-dependent aggregate formation was seen in TBP/Q61~79-GFP cells, whereas no aggregate was seen in TBP/Q36-GFP cells (Figure 3C). When neuronal phenotype was examined after 7~21 days of differentiation, significantly less process and branch in TBP/Q79-GFP cells was observed at 2~3 weeks of differentiation compared with TBP/Q36-GFP cells (1.63~2.12 versus 1.72~2.38 for process [P=0.000~0.002]; 0.37~1.03 versus 0.43~1.31 for branch [P=0.043~0.009]) (Figure 3D).


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

Kung PJ, Tao YC, Hsu HC, Chen WL, Lin TH, Janreddy D, Yao CF, Chang KH, Lin JY, Su MT, Wu CH, Lee-Chen GJ, Hsieh-Li HM - Drug Des Devel Ther (2014)

SH-SY5Y cells with induced TBP/Q36~79-GFP expression and neuronal phenotype.Notes: (A) Real-time polymerase chain reaction quantification (n=3) of TBP/Q36~79-GFP mRNA level relative to HPRT1 mRNA after 2 days of induction with doxycycline (+ Dox) or not (− Dox). (B) Western blot analysis of TBP/Q36~79-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/Q36~79-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/Q36~79-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.
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f3-dddt-8-1929: SH-SY5Y cells with induced TBP/Q36~79-GFP expression and neuronal phenotype.Notes: (A) Real-time polymerase chain reaction quantification (n=3) of TBP/Q36~79-GFP mRNA level relative to HPRT1 mRNA after 2 days of induction with doxycycline (+ Dox) or not (− Dox). (B) Western blot analysis of TBP/Q36~79-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/Q36~79-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/Q36~79-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.
Mentions: To test the aggregation reduction potential of indole and NC001-8 in neuronal cells, we constructed Flp-In SH-SY5Y SCA17 cells with N-terminal TBP/Q36~79-GFP expression in an inducible fashion. As shown in Figure 3A, real-time polymerase quantification of these TBP lines shows 9~11 times TBP expression after induction with doxycycline for 2 days. In immunoblot, TBP antibody detected 52~62 kDa TBP/Q36~79-GFP protein in addition to the endogenous 43 kDa TBP protein (Figure 3B). When TBP/Q36~79 SH-SY5Y cells were differentiated for 7 to 21 days, using retinoic acid,39,40 a Q length-dependent and expression time-dependent aggregate formation was seen in TBP/Q61~79-GFP cells, whereas no aggregate was seen in TBP/Q36-GFP cells (Figure 3C). When neuronal phenotype was examined after 7~21 days of differentiation, significantly less process and branch in TBP/Q79-GFP cells was observed at 2~3 weeks of differentiation compared with TBP/Q36-GFP cells (1.63~2.12 versus 1.72~2.38 for process [P=0.000~0.002]; 0.37~1.03 versus 0.43~1.31 for branch [P=0.043~0.009]) (Figure 3D).

Bottom Line: The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events.We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells.Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.

ABSTRACT
In spinocerebellar ataxia type 17 (SCA17), the expansion of a translated CAG repeat in the TATA box binding protein (TBP) gene results in a long polyglutamine (polyQ) tract in the TBP protein, leading to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On SH-SY5Y cells with inducible SCA17 TBP/Q79-green fluorescent protein (GFP) expression to test indole and synthetic derivative NC001-8 for neuroprotection. We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells. The effects on promoting neurite outgrowth and on reduction of aggregation on Purkinje cells were also confirmed with cerebellar primary and slice cultures of SCA17 transgenic mice. Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment.

Show MeSH
Related in: MedlinePlus