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Effects of DHA on Hippocampal Autophagy and Lysosome Function After Traumatic Brain Injury

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Abstract

Traumatic brain injury (TBI) triggers endoplasmic reticulum (ER) stress and impairs autophagic clearance of damaged organelles and toxic macromolecules. In this study, we investigated the effects of the post-TBI administration of docosahexaenoic acid (DHA) on improving hippocampal autophagy flux and cognitive functions of rats. TBI was induced by cortical contusion injury in Sprague–Dawley rats, which received DHA (16 mg/kg in DMSO, intraperitoneal administration) or vehicle DMSO (1 ml/kg) with an initial dose within 15 min after the injury, followed by a daily dose for 3 or 7 days. First, RT-qPCR reveals that TBI induced a significant elevation in expression of autophagy-related genes in the hippocampus, including SQSTM1/p62 (sequestosome 1), lysosomal-associated membrane proteins 1 and 2 (Lamp1 and Lamp2), and cathepsin D (Ctsd). Upregulation of the corresponding autophagy-related proteins was detected by immunoblotting and immunostaining. In contrast, the DHA-treated rats did not exhibit the TBI-induced autophagy biogenesis and showed restored CTSD protein expression and activity. T2-weighted images and diffusion tensor imaging (DTI) of ex vivo brains showed that DHA reduced both gray matter and white matter damages in cortical and hippocampal tissues. DHA-treated animals performed better than the vehicle control group on the Morris water maze test. Taken together, these findings suggest that TBI triggers sustained stimulation of autophagy biogenesis, autophagy flux, and lysosomal functions in the hippocampus. Swift post-injury DHA administration restores hippocampal lysosomal biogenesis and function, demonstrating its therapeutic potential.

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Acknowledgements

This work was supported in part by the National Institutes of Health [R01NS089051] (DS) and the US Department of Veterans Affairs VA [RR&D#B6761R] (CED). Y.Y. was supported by the Chinese Dalian Municipal Bureau Study Abroad Research Award.

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Authors and Affiliations

  1. Department of Neurology, The Second Hospital of Dalian Medical University, Dalian, 116027, China

    Yan Yin

  2. Department of Neurology, University of Pittsburgh, Pittsburgh, PA, 15213, USA

    Yan Yin, Eric Li, George Sun, Insiya Y. Attarwala & Dandan Sun

  3. Department of Neurosurgery, Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, PA, 15213, USA

    Hong Q. Yan & C. Edward Dixon

  4. Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Education and Clinical Center, Pittsburgh, PA, 15213, USA

    Hong Q. Yan, C. Edward Dixon & Dandan Sun

  5. Animal Imaging Center, University of Pittsburgh, Pittsburgh, PA, 15213, USA

    Lesley M. Foley & T. Kevin Hitchens

  6. Department of Biological Science, University of Pittsburgh, Pittsburgh, PA, 15213, USA

    Liwia A. Andrzejczuk & Kirill Kiselyov

  7. Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, 15213, USA

    T. Kevin Hitchens

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  1. Yan Yin
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Contributions

Q.H.Y. and C.E.D. conducted CCI. Y.Y. and I.Y.A. performed immunostaining and quantified immunofluorescent images. Y.Y. performed CTSD activity assays. E.L. conducted immunoblotting assays. L.A.A. and K.K. performed and quantified RT-PCR assays. T.K.H. and L.M.F. conducted MRI experiment. Y.Y. and D.S. designed the experiments. Y.Y., G.S., and D.S. completed the manuscript writing.

Corresponding author

Correspondence to Dandan Sun.

Ethics declarations

All studies were in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals from the US Department of Health and Human Services and were approved by the University of Pittsburgh Medical Center Institutional Animal Care and Use Committee.

Conflict of Interest

The authors declare that they have no conflicts of interest.

Electronic Supplementary Material

Supplemental Figure 1.

No changes of CTSD protein expression in CA2 or CA3 regions after TBI. Representative confocal immnunofluorescence images show no changes in CTSD protein expression in CA2 or CA3 regions in the TBI + Vehicle and TBI + DHA rats at 3 days post-TBI. (GIF 302 kb)

High resolution image (TIFF 59225 kb)

Supplemental Figure 2.

No differences of LAMP1 and CTSD protein expression in naive and SHAM hippocampus. Representative confocal immnunofluorescence images show changes in LAMP1 (a) or CTSD (b) protein expression in CA1 regions in the naïve, SHAM, or TBI + Vehicle rats at 3 days post-TBI. Representative Western blots of LAMP1 (c) expression in the CL and IL hippocampi of naïve, Sham, or TBI + Vehicle-treated rats at 3 days after TBI. The same blot was probed with the antibody against β-actin as a loading control. (GIF 385 kb)

High resolution image (TIFF 66624 kb)

Supplemental Figure 3.

Changes of hippocampal protein ubiquitination and microtubule-associated light chain 3 protein after TBI. (a) Representative Western blots of LC3 expression in the CL and IL hippocampi of Sham, TBI + Vehicle, and TBI + DHA-treated rats at 3 days after TBI. The same blot was probed with the antibody against β-actin as a loading control. Summary data expressed as the band intensity ratio between LC3I and β-actin. The values are the mean ± SE (n = 6). * p < 0.05. No significant changes in LC3 were observed in the TBI+ Vehicle and TBI+DHA rats. (b) Representative Western blots of protein ubiquitination in the CL and IL hippocampi of Sham, TBI + Vehicle, and TBI + DHA-treated rats at 3 days TBI. The intensity of proteins in a range of molecular weights from 250 kDa to 50 kDa was calculated. The same blot was probed with the antibody against β-actin as a loading control. Summary data were normalized with β-actin. The values are the mean ± SE (n = 8). **p < 0.01 vs Sham, ***p < 0.001 vs Sham. No significant changes in protein ubiquitination were observed in the TBI+ Vehicle and TBI+DHA rats. (GIF 51 kb)

High resolution image (TIFF 37535 kb)

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Yin, Y., Li, E., Sun, G. et al. Effects of DHA on Hippocampal Autophagy and Lysosome Function After Traumatic Brain Injury. Mol Neurobiol 55, 2454–2470 (2018). https://doi.org/10.1007/s12035-017-0504-8

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  • DOI: https://doi.org/10.1007/s12035-017-0504-8

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