Hsin-Han Yang,1,† I-Tsang Chiang,1,† Jen-Wei Liu,2 Jeanne Hsieh,2 Jui-Hao Lee,2 Huai-En Lu,3 Hwa-Sung Tso,1 Yu-Chen Deng,1 Jo-Chi Kao,1 Jhen-Rong Wu,1 Horng-Jyh Harn,4,5,* and Tzyy-Wen Chiou1,*


"1Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan; [email protected] (H.-H.Y.); [email protected] (I.-T.C.); [email protected] (H.-S.T.); [email protected] (Y.-C.D.); [email protected] (J.-C.K.); [email protected] (J.-R.W.)
2Everfront Biotech Inc., New Taipei City 221, Taiwan; [email protected] (J.-W.L.); [email protected] (J.H.); [email protected] (J.-H.L.)
3Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu 300, Taiwan; [email protected]
4Bioinnovation Center, Buddhist Tzu Chi Foundation, Hualien 970, Taiwan
5Department of Pathology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien 970, Taiwan
*Correspondence: [email protected]_ekud (H.-J.H.); [email protected] (T.-W.C.)
†These authors contributed equally to this work."


Calcium Imaging, Medical Research


"Spinocerebellar ataxia type 3 (SCA3) is characterized by the over-repetitive CAG codon in the ataxin-3 gene (ATXN3), which encodes the mutant ATXN3 protein. The pathological defects of SCA3 such as the impaired aggresomes, autophagy, and the proteasome have been reported previously. To date, no effective treatment is available for SCA3 disease. This study aimed to study anti-excitotoxic effects of n-butylidenephthalide by chemically insulted Purkinje progenitor cells derived from SCA3 iPSCs. We successfully generated Purkinje progenitor cells (PPs) from SCA3 patient-derived iPSCs. The PPs, expressing both neural and Purkinje progenitor’s markers, were acquired after 35 days of differentiation. In comparison with the PPs derived from control iPSCs, SCA3 iPSCs-derived PPs were more sensitive to the excitotoxicity induced by quinolinic acid (QA). The observations of QA-treated SCA3 PPs showing neural degeneration including neurite shrinkage and cell number decrease could be used to quickly and efficiently identify drug candidates. Given that the QA-induced neural cell death of SCA3 PPs was established, the activity of calpain in SCA3 PPs was revealed. Furthermore, the expression of cleaved poly (ADP-ribose) polymerase 1 (PARP1), a marker of apoptotic pathway, and the accumulation of ATXN3 proteolytic fragments were observed. When SCA3 PPs were treated with n-butylidenephthalide (n-BP), upregulated expression of calpain 2 and concurrent decreased level of calpastatin could be reversed, and the overall calpain activity was accordingly suppressed. Such findings reveal that n-BP could not only inhibit the cleavage of ATXN3 but also protect the QA-induced excitotoxicity from the Purkinje progenitor loss.

Keywords: n-butylidenephthalide, SCA3, ATXN3, iPSCs, Purkinje progenitor, quinolinic acid



The cells were placed under an upright widefield epifluorescence microscope (ECLIPSE Ti2, Nikon, Tokyo, Japan) equipped with a 340/380 nm LED system (pE-340fura, CoolLED, Andover, UK).

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The 340 nm and 380 nm LED illumination system provides the optimum excitation wavelengths for Fura-2-based calcium imaging, allowing high-precision, stable, high-throughput imaging with video-rate time resolution.

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International Journal of Molecular Sciences

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