eEF1A2 siRNA Suppresses MPP+-Induced Activation of Akt and mTOR and Potentiates Caspase-3 Activation in a Parkinson's Disease Model.

The tissue-specific protein eEF1A2 has been linked to the development of neurological disorders. The role of eEF1A2 in the pathogenesis of Parkinson's disease (PD) has yet to be investigated. The aim of this study was to determine the potential neuroprotective effects of eEF1A2 in an MPP+ model of PD. Differentiated SH-SY5Y cells were transfected with eEF1A2 siRNA, followed by MPP+ exposure. The expression of p-Akt1 and p-mTORC1 was determined using Western blotting. The expression of p53, Bax, Bcl-2, and caspase-3 was evaluated using qRT-PCR. Cleaved caspase-3 levels and Annexin V/propidium iodide flow cytometry were used to determine apoptosis. The effects of PI3K inhibition were examined. The results showed that eEF1A2 siRNA significantly reduced the eEF1A2 expression induced by MPP+. MPP+ treatment activated Akt1 and mTORC1; however, eEF1A2 knockdown suppressed this activation. In eEF1A2-knockdown cells, MPP+ treatment increased the expression of p53 and caspase-3 mRNA levels as well as increased apoptotic cell death when compared to MPP+ treatment alone. In cells exposed to MPP+, upstream inhibition of the Akt/mTOR pathway, by either LY294002 or wortmannin, inhibited the phosphorylation of Akt1 and mTORC1. Both PI3K inhibitors increased eEF1A2 expression in cells, whether or not they were also treated with MPP+. In conclusion, eEF1A2 may function as a neuroprotective factor against MPP+, in part by regulating the Akt/mTOR pathway upstream.

eEF1A2 knockdown impairs neuronal proliferation and inhibits neurite outgrowth of differentiating neurons.

OBJECTIVES: The translation elongation factor-1, alpha-2 (eEF1A2) plays an important role in protein synthesis. Mutations in this gene have been described in individuals with neurodevelopmental disorders. Here, we silenced the expression of eEFA2 in human SH-SY5Y neuroblastoma cells and observed its roles in neuronal proliferation and differentiation upon induction with retinoic acid. METHODS: eEF1A2 were silenced using siRNA transfection. Cell proliferation was qualitatively evaluated by Ki-67 immunocytochemistry. Neuronal differentiation was induced with retinoic acid for 3, 5, 7 and 10 days. Neurite length was measured. The expression of microtubule-associated protein 2 (MAP2) was analyzed by western blotting. Tyrosine hydroxylase expression was visualized by immunofluorescence. Cytotoxicity to a neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and western blotting of cleaved caspase-3. RESULTS: eEF1A2 knockdown suppressed the proliferative activity of undifferentiated SH-SY5Y cells as shown by decreased Ki-67 immunostaining. Upon retinoic acid-induction, differentiated neurons with eEF1A2 knockdown exhibited shorter neurite length than untransfected cells, which was associated with the reduction of tyrosine hydroxylase and suppression of MAP2 at 10 days of differentiation. eEF1A2 knockdown decreased the survival of neurons, which was clearly observed in undifferentiated and short-term differentiated cells. Upon treatment with MPP+, cells with eEF1A2 knockdown showed a further reduction in cell survival and an increase of cleaved caspase-3 protein. CONCLUSIONS: Our results suggest that eEF1A2 may be required for neuronal proliferation and differentiation of SH-SY5Y cells. Increased cell death susceptibility against MPP+ in eEF1A2-knockdown neurons may imply the neuroprotective role of eEF1A2.

Effects of eEF1A2 knockdown on autophagy in an MPP+-induced cellular model of Parkinson's disease.

1-Methyl-4-phenylpyridinium ion (MPP+) is widely used to induce a cellular model of Parkinson's disease (PD) in dopaminergic cell lines. Downregulation of the protein translation elongation factor 1 alpha (eEF1A) has been reported in the brain tissue of PD patients. eEF1A2, an isoform of eEF1A, is associated with lysosome biogenesis that involves the autophagy process. However, the role of eEF1A2 on autophagic activity in PD has not been elucidated. In this work, we investigated the role of eEF1A2 on autophagy using eEF1A2 siRNA knockdown in differentiated SH-SY5Y neuronal cells treated with MPP+. We found that eEF1A2 was upregulated in differentiated cells, which could be silenced by eEF1A2 siRNA. Significantly, cells treated with MPP+ after eEF1A2 knockdown showed a decreased number of LC3 puncta, decreased LC3-II/LC3-I ratio, and decreased phospho-Beclin-1, compared to the MPP+ alone group. These cells showed extensive areas of mitochondria damage, with a reduction of mitochondrial membrane potential, but reduced mitophagy as indicated by the reduced colocalization of LC3 puncta with damaged mitochondria. Cells with eEF1A2 siRNA plus MPP+ treatment aggravated α-synuclein accumulation but reduced colocalization with LC3. As a result, eEF1A2 knockdown decreased viability, increased apoptotic nuclei, increased caspase-3/7 activation and increased cleaved caspase-3 when cells were treated with MPP+. These results suggest that eEF1A2 is essential for dopaminergic neuron survival against MPP+, in part through autophagy regulation.

Inhibition of the antioxidant enzyme PRDX1 activity promotes MPP+-induced death in differentiated SH-SY5Y cells and may impair its colocalization with eEF1A2.

AIM: eEF1A2 is highly expressed in postmitotic cells and has been reported to interact with the antioxidant enzyme peroxiredoxin 1 (PRDX1). PRDX1 is involved in motor neuron differentiation. Here, we studied the relationship between eEF1A2 and PRDX1 during dopaminergic neuron differentiation, and examined their possible association in an oxidative stress model of Parkinson's disease (PD). MAIN METHODS: Expression of eEF1A2 and PRDX1 in SH-SY5Y cells at various durations of retinoic acid (RA) induction was detected using qRT-PCR, Western blotting and immunofluorescence. Neurons of 10-day differentiation were treated with the PRDX1 inhibitor H7, MPP+ and H7 plus MPP+. The cell viability, the amounts of apoptotic nuclei, DHE signals, and the expression of p53, p-Akt and p-mTOR were determined. The colocalization of eEF1A2 and PRDX1 was visualized using confocal microscopy. KEY FINDINGS: eEF1A2 gradually increased after RA-induced differentiation of SH-SY5Y cells, while PRDX1 protein gradually decreased. MPP+ treatment increased eEF1A2 in both undifferentiated and differentiated neurons; however, PRDX1 appeared to elevate only in mature neurons. The inhibition of the PRDX1 activity with H7 promoted MPP+-induced cell death, as evidenced by decreased cell viability, increased apoptotic nuclei, increased the DHE signal, and increased p53. However, H7 induced the activation of the prosurvival Akt and mTOR in MPP+-treated cells. Besides, a colocalization of eEF1A2 and PRDX1 was evidenced in MPP+-treated neurons. This colocalization was possibly prevented by inhibiting the PRDX1 activity, resulting in aggravated neuronal death. SIGNIFICANCE: Our results suggest that the possible association between eEF1A2 and PRDX1 may be a promising target for modifying neuronal death in PD.