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. 2019 Jun;34(3):715-720.
doi: 10.1007/s11011-019-00405-4. Epub 2019 Mar 9.

Genistein Induces Degradation of Mutant Huntingtin in Fibroblasts From Huntington's Disease Patients

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Free PMC article

Genistein Induces Degradation of Mutant Huntingtin in Fibroblasts From Huntington's Disease Patients

Karolina Pierzynowska et al. Metab Brain Dis. .
Free PMC article

Abstract

Mutations in the HTT gene, consisting of expansion of CAG triplets, cause the Huntington's disease (HD), one of the major neurodegenerative disorders. Formation of aggregates of mutant huntingtin (mHTT, the product of the mutant HTT gene) leads to cellular dysfunctions, and subsequent neurodegeneration which manifest clinically as motor abnormalities and cognitive deficits. We recently used immortalized HEK-293 cells expressing the 1st exon of the mutant HTT gene as a cellular model of HD, and showed that the stimulation of autophagy by genistein corrected the mutant phenotype. However, effects of genistein on HD patient-derived cells remained unknown. In this report, we demonstrated that genistein also instigated degradation of mHTT in fibroblasts derived from HD patients. This was assessed as a significant decrease in the levels of HTT in HD fibroblasts measured by Western-blotting, and the disappearance of intracellular mHTT aggregates in cells observed by fluorescent microscopy. Fibroblasts derived from control persons were not affected by genistein treatment. These results indicate that genistein can improve HD phenotype in patient-derived cells, and substantiates the need for further studies of this isoflavone as a potential therapeutic agent.

Keywords: Autophagy; Genistein; Huntingtin; Huntington’s disease; Protein aggregates; Protein degradation.

Conflict of interest statement

The use of genistein in treatment of Huntington’s disease is a subject of patent application (no. P.417983, submitted to Polish Patent Office). No other conflicts of interest are declared by the authors.

Figures

Fig 1
Fig 1
Genistein reduces the number of mHTT aggregates in human fibroblasts. Cultures of fibroblasts derived from four patients suffering from Huntington’s disease (HD) and four healthy persons (WT) were treated with either DMSO (control) or 30, 60 and 100 μM genistein, for 48 h. Cells were immuno-stained with anti-huntingtin antibody, secondary antibody conjugated with Alexa Fluor 488, and analyzed by fluorescent microscopy at 1000 x magnification. The number of aggregates per 100 μm2 were counted in 100 cells per culture. The experiments were performed in triplicates. Panel A demonstrates representative cells from each group. Panel B shows quantitative analysis of the number of aggregates, where bars represent mean values ± SD, and asterisks indicate statistically significant differences (p < 0.05) between WT and HD groups. Panel C demonstrates association of mHTT with cytoskeletal fibers in fibroblasts derived from HD patient. In panels A and C, white scale bars indicate 10 μm
Fig 2
Fig 2
Genistein normalizes levels of HTT in HD fibroblasts. Cultures of fibroblasts derived from four HD patients (HD) and four healthy persons (WT) were treated with either DMSO (control) or 30, 60 and 100 μM genistein, for 48 h. Cellular proteins were separated and detected using the WES system. Four HD and four WT cell lines were used, and the analyses were performed in triplicates. Representative blots are shown in the upper panel, and quantitative analysis is presented in the lower panel. Bars represent mean values ± SD from three independent experiments. Asterisks indicate statistically significant differences (p < 0.05) between WT control and HD groups

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