Downregulation of α-Synuclein Protein Levels by an Intracellular Single-Chain Antibody

Article type: Research Article

Authors: Chen, Yun-Hsiang^{a; *} | Yu, Seong-Jin^b | Wu, Kuo-Jen^b | Wang, Yu-Syuan^b | Tsai, Ho-Min^b | Liao, Li-Wen^a | Chen, Shuchun^{b; c} | Hsieh, Wei^{a; b} | Chen, Hsi^b | Hsu, Shu-Ching^{d; e; f} | Chen, Mao-Liang^g | Hoffer, Barry J.^h | Wang, Yun^b

Affiliations: [a] Department of Life Science, Fu-Jen Catholic University, New Taipei City, Taiwan | [b] Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Taiwan | [c] Graduate Institute of Applied Science and Engineering, Fu-Jen Catholic University, New Taipei City, Taiwan | [d] Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan, Taiwan | [e] Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan | [f] PhD Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University, Taichung City, Taiwan | [g] Department of Research, Taipei Tzu Chi Hospital, Buddhist Tzy Chi Medical Foundation, New Taipei City, Taiwan | [h] Department of Neurosurgery, Case Western Reserve University, School of Medicine, Cleveland, OH, USA

Correspondence: [*] Correspondence to: Yun-Hsiang Chen, Fu-Jen Catholic University, Department of Life Science, No. 510 Zhungzheng Rd., Xinzhuang Dist., New Taipei City, 24205, Taiwan. Tel.: +886 2 2905 2462; Fax: +886 2 2905 2193; E-mail: [email protected].

Abstract: Background:Accumulation of α-synuclein (αSyn) in the dopaminergic neurons is a common pathology seen in patients with Parkinson’s disease (PD). Overproduction of αSyn potentiates the formation of oligomeric αSyn aggregates and enhances dopaminergic neuron degeneration. Downregulating intracellular monomeric αSyn prevents the formation of αSyn oligomers and is a potential therapeutic strategy to attenuate the progression of PD. Objective:The purpose of this study is to investigate the efficacy of gene delivery of αSyn-specific single-chain antibodies in vitro and in vivo. Methods and Results:The plasmids for αSyn and selective antibodies (NAC32, D10, and VH14) were constructed and were transfected to HEK293 and SH-SY5Y cells. Co-expression of αSyn with NAC32, but not D10 or VH14, profoundly downregulated αSyn protein, but not αSyn mRNA levels in these cells. The interaction of αSyn and NAC32 antibody was next examined in vivo. Adeno-associated virus (AAV)-αSyn combined with AAV-NAC32 or AAV-sc6H4 (a negative control virus) were stereotactically injected into the substantia nigra of adult rats. AAV-NAC32 significantly reduced AAV-encoded αSyn levels in the substantia nigra and striatum and increased tyrosine hydroxylase immunoreactivity in the striatum. Also, in the animals injected with AAV-NAC32 alone, endogenous αSyn protein levels were significantly downregulated in the substantia nigra. Conclusion:Our data suggest that AAV-mediated gene transfer of NAC32 is a feasible approach for reducing the expression of target αSyn protein in brain.

Keywords: Adeno-associted virus, intrabody, Parkinson’s disease, alpha synuclein, tyrosine hydroxylase

DOI: 10.3233/JPD-191787

Journal: Journal of Parkinson's Disease, vol. 10, no. 2, pp. 573-590, 2020