Generation and characterisation of leucine-rich repeat kinase 2 (LRRK2) knockout sh-sy5y neuroblastoma cells and investigation of the effects of edible bird's nest extarct

Jong, Hui Lan (2026) Generation and characterisation of leucine-rich repeat kinase 2 (LRRK2) knockout sh-sy5y neuroblastoma cells and investigation of the effects of edible bird's nest extarct. PhD thesis, UTAR.

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Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major genetic contributor to Parkinson’s disease (PD), with pathogenic variants increasing kinase activity and motivating LRRK2 inhibition as a therapeutic strategy. However, abnormal phenotypes in LRRK2 knockout models raise safety concerns. Emerging evidence also implicates LRRK2 in cancer biology, with pathogenic mutations associated with increased malignancy risk, prompting investigation of LRRK2 inhibition in oncology. The human neuroblastoma cell line SH-SY5Y is widely used in PD research but expresses LRRK2 at low levels, presenting challenges for gene knockout studies. In this study, an optimised strategy was developed to generate LRRK2 knockout SH-SY5Y (LKO) cells. A double-cut CRISPR/Cas9 system with multiple guide RNAs was employed, alongside optimisation of electroporation conditions, clonal expansion procedures, and sensitive protein detection methods. Successful knockout of LRRK2 was confirmed by PCR, Sanger sequencing, and Western blot analysis. Phenotypic characterisation of LKO cells revealed marked morphological and functional alterations, including changes in cell morphology, adhesion, proliferation, senescence, autophagy, and mitochondrial respiration. LKO cells underwent conversion into intermediate (I-type) and substrate-adherent/Schwannian (S-type) neuroblastoma phenotypes and displayed hallmarks of cellular senescence, such as enlarged and flattened morphology, loss of neurite-like processes, increased substrate adhesion, reduced proliferation, elevated senescence-associated β-galactosidase activity, and decreased autophagic activity and mitochondrial respiration. Among the LKO clones, LKO1 (I-type) retained the ability to differentiate into N-type cells and dopaminergic neurons, making it suitable for PD-related studies and a potential model for investigating LRRK2-associated cancer stem cell-like properties. In contrast, LKO2 and LKO3 (S-type) provided insights into neuroblastoma cell plasticity. Using LKO1 cells, the effects of edible bird’s nest (EBN) acid extract were evaluated. The extract exhibited antioxidant activity but significantly stimulated cell proliferation and autophagy, suggesting that EBN may promote senescence evasion and support cancer cell growth. Despite its antioxidant properties, EBN may therefore be unsuitable as a complementary therapy for cancer patients. Keywords: leucine-rich repeat kinase 2; knockout; neuroblastoma; edible bird’s nest; senescence Subject Area: RC321-571 Neurosciences

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