Relevance of AIF/CypA Lethal Pathway in SH-SY5Y Cells Treated with Staurosporine.

Mariarosaria Conte,Rosanna Palumbo,Lucia Altucci,Alessandra Monti,Angela Nebbioso,Nunzianna Doti,Menotti Ruvo,Elisabetta Fontana

doi:10.3390/ijms23010265

Abstract

The AIF/CypA complex exerts a lethal activity in several rodent models of acute brain injury. Upon formation, it translocates into the nucleus of cells receiving apoptotic stimuli, inducing chromatin condensation, DNA fragmentation, and cell death by a caspase-independent mechanism. Inhibition of this complex in a model of glutamate-induced cell death in HT-22 neuronal cells by an AIF peptide (AIF(370-394)) mimicking the binding site on CypA, restores cell survival and prevents brain injury in neonatal mice undergoing hypoxia-ischemia without apparent toxicity. Here, we explore the effects of the peptide on SH-SY5Y neuroblastoma cells stimulated with staurosporine (STS), a cellular model widely used to study Parkinson’s disease (PD). This will pave the way to understanding the role of the complex and the potential therapeutic efficacy of inhibitors in PD. We find that AIF(370-394) confers resistance to STS-induced apoptosis in SH-SY5Y cells similar to that observed with CypA silencing and that the peptide works on the AIF/CypA translocation pathway and not on caspases activation. These findings suggest that the AIF/CypA complex is a promising target for developing novel therapeutic strategies against PD.

Highlights

Parkinson’s disease (PD) is a devastating neurodegenerative disorder for which only symptomatic treatments are available
Complex on myocyte death in arrhythmogenic cardiomyopathy, significantly expanding to other diseases the potential impact of targeting this complex for therapeutic approaches [32]. In this scenario, using apoptosis-inducing factor (AIF)(370-394) as a prototypical inhibitor, we have investigated the possible crosstalk between the AIF/cyclophilin A (CypA) complex and STS-evoked cell death in MTT and flow cytometry assays have been used to assess cell viability and apoptosis, whereas the associated molecular mechanism has been assessed by Western blotting (WB)
FACS analyses of apoptotic cells stained with Propidium Iodide (PI) were3 of 12 performed, showing that STS treatment led to a significant percentage of PI positive cells (~60%) already at 1 μM with an increase up to 75% at 20 μM (Figure 1D)

Summary

Introduction

Parkinson’s disease (PD) is a devastating neurodegenerative disorder for which only symptomatic treatments are available. SH-SY5Y cells can be treated with staurosporine (STS), a protein kinase inhibitor, which provokes cell death through both caspase-dependent and independent pathways [2,3,4]. SH-SY5Y cells treated with high concentrations of STS (over 0.5 μM) do not die following a characteristic necrotic phenotype but rather due to oxidative damage. Consistent with this idea, in the presence of high concentrations of STS, caspase inhibition by z-VAD-fmk, a broad-spectrum caspase inhibitor, reduces the apoptotic phenotype but does not inhibit cell death, which instead appears to be due to oxidative damage [5]. It has been demonstrated that STS treatment provides the nuclear translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus, where it exerts a proapoptotic activity [7,9,10]

Methods

Results

Conclusion