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Published on: May 7, 2014
HspB5 protects mouse neural stem/progenitor cells from paraquat toxicity
Naveen Kumar Mekala1, Shyama Sasikumar2, Kranthi Kiran Akula3
1College of Medicine, Central Michigan University Mt Pleasant, MI 48858, USA.
Introduction:
HspB5 (αB-crystallin) is known to be involved in a variety of cellular functions, including, protection of cells from oxidative damage and inhibiting apoptosis. Neural stem/progenitor cells (NSPCs) have significant therapeutic value, especially in the NSC/NPC transplantation therapy. However, the viability of the transplanted NSPCs remains low because of various factors, including oxidative stress.
Objective:
The current investigation explored the possible role of HspB5 in the protection of mouse NSPCs (mNSPCs) against paraquat-induced toxicity.
Methods:
The recombinant human HspB5 was expressed in E.coli and was purified using gel filtration and Ion-exchange chromatography. The biophysical characterization of HspB5 was carried out using DLS, CD, and Analytical Ultracentrifugation (SV); the chaperone activity of HspB5 was determined by alcohol dehydrogenase aggregation assay. We have subjected the mNSPCs to paraquat-induced oxidative stress and monitored the protective ability of HspB5 by MTT assay and Hoechst-PI staining. Furthermore, increase in the expression of the anti-apoptotic protein, procaspase-3 was monitored using western blotting.
Results:
The recombinant HspB5 was purified to its homogeneity and was characterized using various biophysical techniques. The externally added FITC-labeled HspB5 was found to be localized within the cytoplasm of mNSPCs. Our Immunocytochemistry results showed that the externally added FITC-labeled HspB5 not only entered the cells but also conferred cytoprotection against paraquat-induced toxicity. The protective events were monitored by a decrease in the PI-positive cells and an increase in the procaspase-3 expression through Immunocytochemistry and Western blotting respectively.
Conclusion:
Our results clearly demonstrate that exogenously added recombinant human HspB5 enters the mNSPCs and confers protection against paraquat toxicity.
Insights
Recombinant human heat shock protein B5 (HspB5) protects mouse neural stem/progenitor cells (mNSPCs) from oxidative stress. Exogenously added HspB5 enters mNSPCs, reducing toxicity and enhancing cell viability.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Heat shock protein B5 (HspB5), also known as αB-crystallin, plays a role in cellular protection against oxidative damage and apoptosis.
- Neural stem/progenitor cells (NSPCs) are crucial for regenerative therapies, but their survival is compromised by oxidative stress.
- Transplanted NSPCs exhibit low viability due to factors like oxidative stress, highlighting the need for protective strategies.
Purpose of the Study:
- To investigate the protective role of HspB5 in mouse NSPCs (mNSPCs) against paraquat-induced toxicity.
- To determine if exogenous HspB5 can confer cytoprotection to mNSPCs under oxidative stress conditions.
Main Methods:
- Recombinant human HspB5 was expressed, purified, and characterized using biophysical techniques (DLS, CD, AUC).
- Chaperone activity of HspB5 was assessed via alcohol dehydrogenase aggregation assay.
- mNSPCs were exposed to paraquat-induced oxidative stress, and HspB5's protective effects were evaluated using MTT assay, Hoechst-PI staining, and Western blotting for procaspase-3 expression.
Main Results:
- Purified recombinant HspB5 exhibited expected biophysical characteristics and chaperone activity.
- FITC-labeled HspB5 successfully entered the cytoplasm of mNSPCs.
- Exogenous HspB5 conferred significant cytoprotection against paraquat-induced toxicity, evidenced by reduced PI-positive cells and increased procaspase-3 expression.
Conclusions:
- Exogenously administered recombinant human HspB5 effectively enters mouse neural stem/progenitor cells.
- HspB5 provides significant protection to mNSPCs against paraquat-induced oxidative stress.
- These findings support HspB5 as a potential therapeutic agent for enhancing NSPC survival in transplantation therapies.
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