Nilotinib as a Prospective Treatment for Alzheimer's Disease: Effect on Proteins Involved in Neurodegeneration and Neuronal Homeostasis

Abstract

Nilotinib, a tyrosine kinase inhibitor that targets the Abelson tyrosine kinase (c-Abl) signaling pathway, is FDA-approved to treat chronic myeloid leukemia. Nilotinib has properties indicative of a possible utility in neuroprotection that have prompted exploration of repurposing the drug for the treatment of Alzheimer's disease (AD) and Parkinson's disease (PD). AD is a progressive age-related neurodegenerative disorder characterized by the deposition of extracellular amyloid-β plaques and intracellular neurofibrillary tangles. It is incurable and affects approximately 50 million patients worldwide. Nilotinib reduces c-Abl phosphorylation, amyloid-β levels, and dopaminergic neuron degeneration in preclinical AD models. This study explores the effects of nilotinib on amyloid processing and mitochondrial functioning in the SH-SY5Y human neuroblastoma cell line. SH-SY5Y cells were exposed to nilotinib (1, 5, and 10 µM). Real-time PCR and immunoblot analysis were performed to quantify the expression of genes pertaining to amyloid-β processing and neuronal health. Nilotinib did not significantly change APP, BACE1, or ADAM10 mRNA levels. However, BACE1 protein was significantly increased at 1 µM, and ADAM10 was increased at 10 µM nilotinib without affecting APP protein expression. Further, nilotinib treatment did not affect the expression of genes associated with neuronal health and mitochondrial functioning. Taken together, our findings do not support the efficacy of nilotinib treatment for neuroprotection.

Keywords: Alzheimer’s disease; amyloid beta; mitochondria; nilotinib; tyrosine kinase inhibitor.

Figure 1

Effect of nilotinib treatment on APP, BACE1, and ADAM10 expression in SH-SY5Y cells. (A–C) Real-time PCR analysis of APP, BACE1, and ADAM10 in SH-SY5Y cells after nilotinib treatment. GAPDH was used as an internal control. (D) Western blot analysis of APP, BACE1, and ADAM10 in SH-SY5Y cells after nilotinib treatment. Densitometry of representative blot was normalized with β-actin. Data were represented in fold difference. N = 6–9, ** p < 0.01, * p < 0.05 based on a one-way ANOVA followed by Bonferroni multi-comparison tests.

Figure 2

Effect of nilotinib treatment on expression of genes involved in neuronal health. (A,B) Real-time PCR analysis of synaptophysin and BDNF in SH-SY5Y cells after nilotinib treatment. GAPDH was used as an internal control. (C) Western blot analysis of synaptophysin in SH-SY5Y cells after nilotinib treatment. Densitometry of representative blot was normalized with β-actin. Data were represented in fold difference. N = 6–9, * p < 0.05, based on a one-way ANOVA followed by Bonferroni multi-comparison tests.

Figure 3

Effect of nilotinib treatment on expression of genes associated with mitochondrial functioning. (A,B) Real-time PCR analysis of TFAM and NRF1 in SH-SY5Y cells after nilotinib treatment. GAPDH was used as an internal control. (C) Western blot analysis of TFAM in SH-SY5Y cells after nilotinib treatment. Densitometry of representative blot was normalized with β-actin. (D) Microscopic images of live SH-SY5Y cells stained with 250 µM of MitoTracker dye after nilotinib treatment. Scale bar: 50 µm. Data were represented in fold difference. N = 6–9, significance was calculated using one-way ANOVA followed by Bonferroni multi-comparison tests.

Figure 4

Effect of nilotinib treatment on mitochondrial morphology. (A–D) Representative TEM images of SH-SY5Y cells treated with 0, 5, and 10 µM concentrations of nilotinib for 48 h. All images were taken at 30,000× magnification. Scale bar: 0.5microns (UM).