Development of ATP13A2-deficient In vitro Model for PARK9 Parkinson’s Disease

Yiing Jye Yap, MD Ezharul Hoque Chowdhury, Rhun Yian Koh, Soi Moi Chye, Kenny Gah Leong Voon, Iekhsan Othman, Khuen Yen Ng

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Abstract

Background: PARK9 familial Parkinson’s disease (PD) is caused by a loss-of-function mutation in the ATP13A2 gene in which the mutation impairs the autophagic-lysosomal degradation pathway and induces intraneuronal accumulation of alpha-synuclein. RNA interference has been a useful tool in generating in vitro knockdown model to study the physiological role of the gene. How-ever, the availability of a validated ATP13A2-deficient in vitro model is limited. Objective: This study aimed to develop the ATP13A2-deficient PD model by delivering ATP13A2 siRNA into neuroblastoma cells using carbonate apatite nanoparticles (CA NPs). Method: CA NPs were fabricated using different concentrations of calcium chloride and character-ised in the presence or absence of ATP13A2 siRNA. Time-dependent stabilities of CA NPs and CA NPs-associated siRNA (CA-siRNA) complex were evaluated by pH, turbidity, size, and zeta potential measurements. The dissolution abilities at acidic conditions of both complexes were investigated. Following that, green fluorescence protein (GFP) and four different siRNAs targeting ATP13A2 (siRNA_5, 6, 7, and 8) were transfected to cells with the fabricated CA NPs. Western blot was per-formed to determine the knockdown effect of the four siRNAs. Results: It was found that 4 mM calcium chloride was ideal for CA NP formation, while an incuba-tion time of 48 hours was required to maintain the stability of nanoparticles. Successful transfection was confirmed by detection of fluorescence signal from the GFP plasmid and the subsequent silenc-ing of this signal by transfecting GFP siRNA. Western blot analysis revealed that ATP13A2 protein expression was significantly reduced to 20% upon transfection with 20 nM of siRNA_5. Conclusion: ATP13A2-deficient PD model was successfully developed.

Original languageEnglish
Pages (from-to)280-291
Number of pages12
JournalCurrent Signal Transduction Therapy
Volume16
Issue number3
DOIs
Publication statusPublished - Dec 2021

Keywords

  • ATP13A2
  • carbonate apatite
  • PARK9
  • Parkinson’s disease
  • siRNA
  • transfection

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