Title:Development of ATP13A2-deficient In vitro Model for PARK9 Parkinson’s Disease
Volume: 16
Issue: 3
Author(s): Yiing Jye Yap, Md Ezharul Hoque Chowdhury, Rhun Yian Koh, Soi Moi Chye, Kenny Gah Leong Voon, Iekhsan Othman and Khuen Yen Ng*
Affiliation:
- School of Pharmacy, Monash University Malaysia, Bandar Sunway, Selangor,Malaysia
Keywords:
Parkinson's disease, ATP13A2, PARK9, siRNA, carbonate apatite, transfection.
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. However,
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 characterised
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 performed
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 incubation
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 silencing
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.
