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Deletion of Nuclear Localizing Signal Attenuates Proinflammatory Activity of Prothymosin-Alpha and Enhances Its Neuroprotective Effect on Transient Ischemic Stroke

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Abstract

Post-ischemic inflammation plays an important role in the progression of ischemia/reperfusion injuries. Prothymosin-α (ProT) can protect cells from necrotic death following ischemia; however, its immunostimulatory actions may counteract the neuroprotective effect. We proposed that ProTΔNLS, synthesized by deleting its nuclear localizing signal (NLS) at the C-terminal of ProT, can attenuate the immunostimulatory activity and has more salient neuroprotective effect. In this study, we examined the therapeutic effects of ProT and ProTΔNLS in a transient middle cerebral artery occlusion (tMCAO) model of rats. Rats that had sustained 90 min of tMCAO were treated with GST-vehicle, ProT, or ProTΔNLS. Therapeutic outcomes were evaluated by infarction volume assay and behavioral assessment. Changes to inflammatory mediators, including tumor necrosis factor α (TNF-α), interleukin-10 (IL-10), and myeloperoxidase (MPO) were evaluated by enzyme-linked immunosorbent assay. Activated matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) levels were evaluated by gelatin zymography. Microglial activation was identified by double-immunostaining for Iba-1 and CD68. Our results showed that while both ProT and ProTΔNLS reduce infarction volume and improve functional outcome, ProTΔNLS provides the best therapeutic outcome. ProT increases TNF-α but decreases IL-10 secretion after ischemic injury, reflecting its pro-inflammatory activity. ProTΔNLS suppresses expression of TNF-α, MPO, and activity of MMPs in ischemic brain tissue. It also suppresses activation of microglia in penumbral cortex. These data demonstrate the immunesuppressive activities of ProTΔNLS. In conclusion, ProT has pro-inflammatory effect that may counteract its neuroprotective effect. Deletion of NLS from ProT may attenuate post-ischemic inflammation and enhance the neuroprotective effects of ProT.

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Acknowledgments

This study was supported by grants from the Ministry of Science and Technology, Taiwan (NSC-102-2314-B-006-038, NSC-103-2314-B-006-053 and NSC-102-2320 -B-006-040-MY3), and the National Cheng Kung University Hospital, Taiwan (NCKUH-10203019 and NCKUH-10407013). The authors thank Ya-Chun Hsiao for the services on image acquisition and analysis from the FACS-like Tissue Cytometry in the Center of Clinical Medicine, National Cheng Kung University Hospital.

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Authors and Affiliations

  1. Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan

    Liang-Chao Wang, Ya-Yun Cheng & Kuen-Jer Tsai

  2. Division of Neurosurgery, Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan

    Liang-Chao Wang

  3. Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan

    Chao-Liang Wu

  4. Center of Clinical Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan

    Kuen-Jer Tsai

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Wang, LC., Wu, CL., Cheng, YY. et al. Deletion of Nuclear Localizing Signal Attenuates Proinflammatory Activity of Prothymosin-Alpha and Enhances Its Neuroprotective Effect on Transient Ischemic Stroke. Mol Neurobiol 54, 582–593 (2017). https://doi.org/10.1007/s12035-015-9671-7

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