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Neuroprotective Efficacy of Betulinic Acid Hydroxamate, a B55α/PP2A Activator, in Acute Hypoxia–Ischemia-Induced Brain Damage in Newborn Rats

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Abstract

There is an increasing evidence of the neuroprotective effects of hypoxia inducing factor prolyl-hydroxylase inhibitors (HIF-PHDi) after hypoxic-ischemic (HI) brain damage (HIBD). We studied the neuroprotective effects of betulinic hydroxamate (BAH), a novel B55α/PP2A activator that dephosphorylates and inhibits PHD2 activity, in a rat model of neonatal HIBD. Seven-day-old (P7) Wistar rats were exposed to hypoxia after left carotid artery electrocoagulation and then received vehicle (HI + VEH) or BAH 3 mg/kg i.p. 30 min post-insult. Brain damage was assessed by magnetic resonance imaging (MRI) and neurobehavioral studies testing motor and cognitive performance at P14 and P37, as well as immunohistochemical studies (TUNEL and myelin basic protein (MBP) signal) at P37. Mechanisms of damage were assessed at P14 determining excitotoxicity (glutamate/N-acetylaspartate ratio by H+-magnetic resonance spectroscopy), oxidative stress (protein nitrosylation by Oxyblot), and inflammation (cytokine and chemokine concentration). BAH reduced brain damage volume and cell death, preventing the development of motor and working memory deficits. BAH showed a robust protective effect on myelination, restoring MBP expression at P37. BAH modulated excitotoxicity, oxidative stress, and inflammation. Most neuroprotective effects were still present despite BAH administration was delayed for 12 h, whereas beneficial effects on motor strength at P14 and on cell death and myelination at P37 were preserved even when BAH administration was delayed for 24 h. In conclusion, BAH appears as an effective neuroprotective treatment for neonatal HIBD in a manner associated with the modulation of excitotoxicity, oxidative stress, and inflammation, showing a broad therapeutic window.

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Data Sharing Statement

Deidentified individual participant data (including data dictionaries) will be made available, in addition to study protocols, the statistical analysis plan, and the informed consent form. Data will be made available upon publication to researchers who provide a methodologically sound proposal for use in achieving the approved proposal’s goals. Proposals should be submitted to the corresponding author.

References

  1. Parikh P, Juul SE. Neuroprotective strategies in neonatal brain injury. J Pediatr. 2018;192:22–32.

    Article  PubMed  Google Scholar 

  2. Gonzalez FF, Ferriero DM. Therapeutics for neonatal brain injury. Pharmacol Ther. 2008;120:43–53.

    Article  CAS  PubMed  Google Scholar 

  3. Johnston MV, Fatemi A, Wilson MA, Northington F. Treatment advances in neonatal neuroprotection and neurointensive care. Lancet Neurol. 2011;10:372–82.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Laptook AR, Shankaran S, Tyson JE, Munoz B, Bell EF, Goldberg RN, et al. Effect of therapeutic hypothermia initiated after 6 hours of age on death or disability among newborns with hypoxic-ischemic encephalopathy a randomized clinical trial. JAMA. 2017;318:1550–60.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Fan X, Heijnen CJ, van der Kooij MA, Groenendaal F, van Bel F. The role and regulation of hypoxia-inducible factor-1α expression in brain development and neonatal hypoxic-ischemic brain injury. Brain Res Rev. 2009;31:99–108.

    Article  Google Scholar 

  6. Trollmann R, Gassmann M. The role of hypoxia-inducible transcription factors in the hypoxic neonatal brain. Brain Dev. 2009;31:503–9.

    Article  PubMed  Google Scholar 

  7. Chu HX, Jones NM. Changes in hypoxia-inducible factor-1 (HIF-1) and regulatory prolyl hydroxylase (PHD) enzymes following hypoxic–ischemic injury in the neonatal rat. Neurochem Res. 2016;41:515–22.

    Article  CAS  PubMed  Google Scholar 

  8. Siddiq A, Aminova LR, Ratan RR. Prolyl 4-hydroxylase activity-responsive transcription factors: from hydroxylation to gene expression and neuroprotection. Front Biosci. 2008;13:2875–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chen W, Jadhav V, Tang J, Zhang JH. HIF-1 alpha inhibition ameliorates neonatal brain damage after hypoxic-ischemic injury. Acta Neurochir Suppl. 2008;102:395–9.

    Article  PubMed  Google Scholar 

  10. Min J, Hu J, He M, et al. Vitexin reduces hypoxia e ischemia neonatal brain injury by the inhibition of HIF-1alpha in a rat pup model. Neuropharmacology. 2015;99:38–50.

    Article  CAS  PubMed  Google Scholar 

  11. Vetrovoy O, Rybnikova E. Neuroprotective action of PHD inhibitors is predominantly HIF-1-independent. J Neurochem. 2019;150:645–7.

    Article  CAS  PubMed  Google Scholar 

  12. Li K, Li T, Wang Y, et al. Sex differences in neonatal mouse brain injury after hypoxia-ischemia and adaptaquin treatment. J Neurochem. 2019;150:759–75.

    Article  CAS  PubMed  Google Scholar 

  13. Stetler RA, Leak RK, Gan Y, et al. Preconditioning provides neuroprotection in models of CNS disease: paradigms and clinical significance. Prog Neurobiol. 2014;114:58–83.

    Article  PubMed  Google Scholar 

  14. Di Conza G, TrussoCafarello S, Loroch S, et al. The mTOR and PP2A pathways regulate PHD2 phosphorylation to fine-tune HIF1α levels and colorectal cancer cell survival under hypoxia. Cell Rep. 2017;18:1699–712.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Ehling M, Celus W, Martín-Perez R, et al. B55α/PP2A limits endothelial cell apoptosis during vascular remodeling: a complementary approach To Disrupt Pathological Vessels? Circ Res. 2020;127:707–23.

    Article  CAS  PubMed  Google Scholar 

  16. Taleski G, Sontag E. Protein phosphatase 2A and tau: an orchestrated “Pas de Deux.” FEBS Lett. 2018;592:1079–95.

    Article  CAS  PubMed  Google Scholar 

  17. Prados ME, Correa-Sáez A, Unciti-Broceta J, et al. Betulinic acid hydroxamate is neuroprotective and induces protein phosphatase 2A-dependent HIF-1α stabilization and post-transcriptional dephosphorylation of prolyl hydrolase 2. Neurotherapeutics. 2021. https://doi.org/10.1007/s13311-021-01089-4.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Pazos MR, Cinquina V, Gómez A, et al. Cannabidiol administration after hypoxia-ischemia to newborn rats reduces long-term brain injury and restores neurobehavioral function. Neuropharmacology. 2012;63:776–83.

    Article  CAS  PubMed  Google Scholar 

  19. Ceprián M, Vargas C, García-Toscano L, et al. Cannabidiol administration prevents hypoxia-ischemia-induced hypomyelination in newborn rats. Front Pharmacol. 2019;10:1131.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Ceprián M, Jiménez-Sánchez L, Vargas C, Barata L, Hind W, Martínez-Orgado J. Cannabidiol reduces brain damage and improves functional recovery in a neonatal rat model of arterial ischemic stroke. Neuropharmacology. 2017;116:151–9.

    Article  PubMed  Google Scholar 

  21. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 3rd edition. San DiegoAcademic Press. 1996.

  22. Barata L, Arruza L, Rodríguez M-J, et al. Neuroprotection by cannabidiol and hypothermia in a piglet model of newborn hypoxic-ischemic brain damage. Neuropharmacology. 2019;146:1–11.

    Article  CAS  PubMed  Google Scholar 

  23. Pazos MR, Mohammed N, Lafuente H, et al. Mechanisms of cannabidiol neuroprotection in hypoxic-ischemic newborn pigs: role of 5HT(1A) and CB2 receptors. Neuropharmacology. 2013;71:282–91.

    Article  CAS  PubMed  Google Scholar 

  24. Lafuente H, Pazos MR, Alvarez A, et al. Effects of cannabidiol and hypothermia on short-term brain damage in new-born piglets after acute hypoxia-ischemia. Front Neurosci. 2016;10:323.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Minassi A, Rogati F, Cruz C, et al. Triterpenoid hydroxamates as HIF prolyl hydrolase inhibitors. J Nat Prod. 2018;81:2235–43.

    Article  CAS  PubMed  Google Scholar 

  26. Trollmann R, Richter M, Jung S, Walkinshaw G, Brackmann F. Pharmacologic stabilization of hypoxia-inducible transcription factors protects developing mouse brain from hypoxia-induced apoptotic cell death. Neuroscience. 2014;278:327–42.

    Article  CAS  PubMed  Google Scholar 

  27. Prados ME, García-Martín AG-M, Unciti-Broceta JD, et al. Betulinic acid hydroxamate prevents colonic inflammation and fibrosis in murine models of inflammatory bowel disease. Acta Pharmacol Sin. 2021;42:1124–38.

    Article  CAS  PubMed  Google Scholar 

  28. Adamcio B, Sperling S, Hagemeyer N, Walkinshaw G, Ehrenreich H. Hypoxia inducible factor stabilization leads to lasting improvement of hippocampal memory in healthy mice. Behav Brain Res. 2010;208:80–4.

    Article  CAS  PubMed  Google Scholar 

  29. Mohammed N, Ceprian M, Jimenez L, Pazos MR, Martínez-Orgado J. Neuroprotective Effects of cannabidiol in hypoxic ischemic insult the Therapeutic Window in Newborn Mice. CNS Neurol Disord Drug Targets. 2017;16:102–8.

    Article  CAS  PubMed  Google Scholar 

  30. Iwai M, Stetler RA, Xing J, et al. Enhanced oligodendrogenesis and recovery of neurological function by erythropoietin following neonatal hypoxic/ischemic brain injury Masanori. Stroke. 2010;41:1032–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Janowska J, Sypecka J. Therapeutic strategies for leukodystrophic disorders resulting from perinatal asphyxia: focus on myelinating oligodendrocytes. Mol Neurobiol. 2018;55:4388–402.

    CAS  PubMed  Google Scholar 

  32. Yuen TJ, Silbereis JC, Griveau A, et al. Oligodendrocyte-encoded HIF function couples postnatal myelination and white matter angiogenesis. Cell. 2014;158:383–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Taie S, Ono J, Iwanaga Y, Tomita S, Asaga T, Chujo K, et al. Hypoxia-inducible factor-1α has a key role in hypoxic preconditioning. J Clin Neurosci. 2009;16:1056–60.

    Article  CAS  PubMed  Google Scholar 

  34. Bergeron M, Yu AY, Solway KE, Semenza GL, Sharp FR. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur J Neurosci. 1999;11:4159–70.

    Article  CAS  PubMed  Google Scholar 

  35. Bernaudin M, Nedelec AS, Divoux D, MacKenzie ET, Petit E, Schumann-Bard P. Normobaric hypoxia induces tolerance to focal permanent cerebral ischemia in association with an increased expression of hypoxia-inducible factor-1 and its target genes, erythropoietin and VEGF, in the adult mouse brain. J Cereb Blood Flow Metab. 2002;22:393–403.

    Article  CAS  PubMed  Google Scholar 

  36. Salama M, Mohamed WM. Tau protein as a biomarker for asphyxia: a possible forensic tool? Appl Transl Genom. 2016;9:20–2.

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are indebted to Maria Cruz Rodríguez-Bobada and Pablo González López for their help performing this experiment.

Funding

This project received funding from Emerald Health Biotechnology España S.LU. and Instituto de Salud Carlos III through the project PI19/00927 (Co-funded by European Regional Development Fund/European Social Fund “A way to make Europe”/ “Investing in your future”).

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Authors and Affiliations

  1. Hospital Clínico San Carlos - IdISSC, Madrid, Spain

    Laura Silva, Carlos Vargas, Aaron del Pozo, María Villa, María Martínez, Lourdes Alvarez & José Martínez-Orgado

  2. Emerald Health Biotechnology España S.L.U, Córdoba, Spain

    María Eugenia Prados & Juan Diego Unciti-Broceta

  3. Maimonides Biomedical Research Institute of Córdoba, University of Córdoba, Córdoba, Spain

    Eduardo Muñoz

  4. Department of Cellular Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain

    Eduardo Muñoz

  5. Reina Sofía University Hospital, Córdoba, Spain

    Eduardo Muñoz

  6. Department of Neonatology, Hospital Clínico San Carlos - IdISSC, Prof. Martin Lagos, s/n, 28040, Madrid, Spain

    José Martínez-Orgado

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  1. Laura Silva
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Correspondence to José Martínez-Orgado.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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José Martinez-Orgado has a Research Agreement with Emerald Health Biotechnology España S.L.U. (Córdoba, Spain). The other authors declare no conflict of interest.

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Juan Diego Unciti-Broceta and Jose Martínez-Orgado are equal senior authors.

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Silva, L., Vargas, C., Prados, M.E. et al. Neuroprotective Efficacy of Betulinic Acid Hydroxamate, a B55α/PP2A Activator, in Acute Hypoxia–Ischemia-Induced Brain Damage in Newborn Rats. Transl. Stroke Res. 14, 397–408 (2023). https://doi.org/10.1007/s12975-022-01017-4

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