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Resveratrol Preconditioning Induces Genomic and Metabolic Adaptations within the Long-Term Window of Cerebral Ischemic Tolerance Leading to Bioenergetic Efficiency

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Abstract

Neuroprotective agents administered post-cerebral ischemia have failed so far in the clinic to promote significant recovery. Thus, numerous efforts were redirected toward prophylactic approaches such as preconditioning as an alternative therapeutic strategy. Our laboratory has revealed a novel long-term window of cerebral ischemic tolerance mediated by resveratrol preconditioning (RPC) that lasts for 2 weeks in mice. To identify its mediators, we conducted an RNA-seq experiment on the cortex of mice 2 weeks post-RPC, which revealed 136 differentially expressed genes. The majority of genes (116/136) were downregulated upon RPC and clustered into biological processes involved in transcription, synaptic signaling, and neurotransmission. The downregulation in these processes was reminiscent of metabolic depression, an adaptation used by hibernating animals to survive severe ischemic states by downregulating energy-consuming pathways. Thus, to assess metabolism, we used a neuronal-astrocytic co-culture model and measured the cellular respiration rate at the long-term window post-RPC. Remarkably, we observed an increase in glycolysis and mitochondrial respiration efficiency upon RPC. We also observed an increase in the expression of genes involved in pyruvate uptake, TCA cycle, and oxidative phosphorylation, all of which indicated an increased reliance on energy-producing pathways. We then revealed that these nuclear and mitochondrial adaptations, which reduce the reliance on energy-consuming pathways and increase the reliance on energy-producing pathways, are epigenetically coupled through acetyl-CoA metabolism and ultimately increase baseline ATP levels. This increase in ATP would then allow the brain, a highly metabolic organ, to endure prolonged durations of energy deprivation encountered during cerebral ischemia.

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Acknowledgments

We would like to acknowledge Claudia Sofia Pereira and Tania Arguello for their help with the Seahorse Biosciences experiments and Isabel Saul for her help with animal blinding and injections.

Funding

This work was supported by the NIH/NINDS grants NS45676, NS054147, NS097658, and NS34773 (to M.A.P.P.), the American Heart Association (AHA) predoctoral award 16PRE29170004 (to N.K.), the NIH F31 predoctoral award NS089356-01A1 (to K.B.K.), and the Lois Pope LIFE Foundation Fellowship (to N.K. and K.B.K.)

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Authors and Affiliations

  1. Cerebral Vascular Disease Research Laboratories, University of Miami Leonard M. Miller School of Medicine, Miami, FL, 33136, USA

    Nathalie Khoury, Jing Xu, Samuel D. Stegelmann, Charles W. Jackson, Kevin B. Koronowski, Kunjan R. Dave & Miguel A. Perez-Pinzon

  2. Department of Neurology, University of Miami, Miller School of Medicine, P.O. Box 016960, Miami, FL, 33101, USA

    Nathalie Khoury, Jing Xu, Samuel D. Stegelmann, Charles W. Jackson, Kevin B. Koronowski, Kunjan R. Dave & Miguel A. Perez-Pinzon

  3. Neuroscience Program, University of Miami Leonard M. Miller School of Medicine, Miami, FL, 33136, USA

    Nathalie Khoury, Jing Xu, Charles W. Jackson, Kevin B. Koronowski, Kunjan R. Dave, Juan I. Young & Miguel A. Perez-Pinzon

  4. John P. Hussman Institute for Human Genomics, University of Miami Leonard M. Miller School of Medicine, Miami, FL, 33136, USA

    Juan I. Young

  5. Department of Human Genetics, University of Miami Leonard M. Miller School of Medicine, Miami, FL, 33136, USA

    Juan I. Young

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  1. Nathalie Khoury
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Correspondence to Miguel A. Perez-Pinzon.

Electronic supplementary material

Supplementary Fig. 1

RPC increases protein levels of ARC at the long-term window post RPC in vivo. a Western blot analysis showing protein levels of ARC and ACTB in the cortex of mice 2 weeks post-RPC. b Quantification of the protein levels of ARC relative to ACTB based on the western blot analysis results. (PNG 124 kb)

High resolution image (TIF 316 kb)

Supplementary Fig. 2

Publically available datasets involving the transcriptomic analysis of mice brains after resveratrol administration. a In the study by Barger et al. in 2008, 30-month-old mice were fed 5 mg/kg resveratrol for 14 months after which their neocortex was subject to a microarray analysis. After analyzing their results using the GEO (Gene Expression Omnibus) tool (GEO2R), we identified 700 differentially expressed genes among which the majority of genes including 473 (67.6%) were significantly downregulated and only 227 (32.4%) genes were upregulated. b In a similar study by Thomas et al. in 2013, 8-week-old mice were fed a much higher dose of resveratrol consisting of 50 mg/kg per day for 6 weeks after which their hippocampus was subject to a microarray analysis. Among the 439 differentially expressed genes, strikingly 422 (96.1%) genes were downregulated and only a very small subset of 17 (3.9%) genes was upregulated. The results of these experiments further support the global downregulation in gene expression that we observed in the cortex of mice 2 weeks post-RPC. (PNG 492 kb)

High resolution image (TIF 2215 kb)

Supplementary Fig. 3

Immunofluorescence staining of the neuronal-astrocytic co-cultures at the long-term window post-RPC confirming cellular identity. Representative images showing the neuronal-astrocytic co-culture model stained with DAPI (blue), NeuN (red), MAP2 (magenta), and GFAP (green) at the 6-day time point post-RPC. (DAPI—4′,6-diamidino-2-phenylindole, GFAP—glial fibrillary acidic protein, NeuN—neuronal nuclei, MAP2—microtubule associated protein 2). (PNG 840 kb)

High resolution image (TIF 6056 kb)

Supplementary Fig. 4

Immunofluorescence staining of the neuronal-astrocytic co-cultures at the long-term window post-RPC confirming neuronal abundance. a Representative images showing the neuronal-astrocytic co-culture model stained with DAPI (blue) and NeuN (magenta) after 6 days from Res or Veh treatment. b Bar graph showing the percentage of neuron relative to DAPI based on the immunostaining results (n = 2; one-way ANOVA; ns, not significant). (PNG 1126 kb)

High resolution image (TIF 5187 kb)

Supplementary Table 1

The list of genes identified from the RNA-seq analysis 2 weeks post-RPC (XLSX 1856 kb)

Supplementary Table 2

The list of genes identified from the microarray analysis conducted on the neocortex from Barger et al. 2008 (XLSX 2357 kb)

Supplementary Table 3

The list of genes identified from the microarray analysis conducted on the hippocampus from Thomas et al. 2013 (XLSX 19 kb)

Supplementary Table 4

The list and sequence of primers used in the real-time PCR experiments (XLSX 14 kb)

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Khoury, N., Xu, J., Stegelmann, S.D. et al. Resveratrol Preconditioning Induces Genomic and Metabolic Adaptations within the Long-Term Window of Cerebral Ischemic Tolerance Leading to Bioenergetic Efficiency. Mol Neurobiol 56, 4549–4565 (2019). https://doi.org/10.1007/s12035-018-1380-6

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