Differences in Gene Expression Profiles and Phenotypes of Differentiated SH-SY5Y Neurons Stably Overexpressing Mitochondrial Ferritin.

Anarmaa Mendsaikhan,Shigeko Takeuchi,Ikuo Tooyama,Douglas G Walker

doi:10.3389/fnmol.2018.00470

Anarmaa Mendsaikhan, Shigeko Takeuchi + Show 2 more

Open Access

https://doi.org/10.3389/fnmol.2018.00470

Copy DOI

Abstract

Mitochondrial ferritin (FtMt) is an iron-transport protein with ferroxidase properties localized to mitochondria. Levels are generally low in all tissues, while increasing the expression of FtMt in neuronal-like cells has been shown to be protective. To determine whether FtMt has potential as a therapeutic approach, there remains the question of how much FtMt is protective. To address this issue, we transfected SH-SY5Y neuroblastoma cells with a FtMt expression plasmid and isolated cell lines with stable expression of FtMt at high, medium and low levels. Using these cell lines, we examined effects of FtMt on neuronal phenotype, neuroprotective activity and gene expression profiles. The phenotypic properties of high, medium and low FtMt expressors were compared with native untransfected SH-SY5Y cells after differentiation with retinoic acid to a neuronal phenotype. Overexpression of FtMt, even in low expressing cells, showed significant protection from oxidative stress induced by hydrogen peroxide or cobalt chloride. Higher levels of FtMt expression did not appear to offer greater protection, and did not have toxic consequences to cells, even though there were significantly more aggregated mitochondria in the highest expressing clone. The phenotypes differed between cell clones when assessed by cell growth, neurite outgrowth, and expression of neuronal proteins including those associated with neurodegenerative diseases. Microarray analysis of high, medium and negative FtMt-expressing cells identified different patterns of expression of certain genes associated with oxidative stress and neuronal development, amongst others. Validation of microarray analyses was carried out by real time polymerase chain reaction. The results showed significant differences in expression of thioredoxin-interacting protein (TXNIP) and microsomal glutathione transfer-1 (MGST-1), which can have critical roles in the regulation of oxidative stress. Differences in expression of calcitonin-related polypeptide alpha (CALCA), growth differentiation factor-15 (GDF-15) and secretogranin II (SCG2) were also observed. Our findings indicate that even low levels of increased FtMt expression can be protective possibly by alterations of some oxidative stress-related and growth factor genes, while high levels of expression did not appear to offer greater protection from oxidative stress or induce significant toxicity in cells. These experiments provide supporting data that increasing FtMt might be a feasible strategy for therapeutics in certain neurodegenerative and neurological diseases.

Highlights

Age-associated neurodegenerative diseases, Alzheimer’s disease (AD) and Parkinson’s disease (PD), have proven resistant to effective therapies as the pathological processes are complex and still incompletely understood
A number of studies have demonstrated that increased levels of FtMt in cells have significant protective effect from a range of insults linked to production of reactive oxygen species (ROS), including those associated with neurodegenerative disease mechanisms
Expressed at low levels compared to ubiquitious iron binding proteins such as ferritin, the protective effect would appear to come from its specific localization to mitochondria, the major cellular source of ROS (Nesi et al, 2017)

Summary

Introduction

Age-associated neurodegenerative diseases, Alzheimer’s disease (AD) and Parkinson’s disease (PD), have proven resistant to effective therapies as the pathological processes are complex and still incompletely understood. There have been multiple approaches aimed at slowing down the degenerative processes, but they have primarily focused on inhibiting the formation or promoting the removal of toxic forms of amyloid beta (Aβ) peptide or tau (for AD) or α-synuclein (for PD) (Brundin et al, 2017; Jan et al, 2017). These diseases have significant clinical and pathological differences, one common feature is increased oxidative stress through elevated levels of damaging reactive oxygen species (ROS) (Nesi et al, 2017; Lang and Espay, 2018). The FtMt gene promoter was shown to contain positive regulatory sequences for cyclic-AMP response elementbinding protein (CREB), YYI and SP1 transcription factors, and C/EBPβ, GATA2 and FOXA1 sequences as negative regulators (Guaraldo et al, 2016)

Methods

Results

Discussion

Conclusion