ACE2 gene transgenesis enhances memory of psychophysiological trauma in mouse models of post-traumatic stress disorder

Introduction. The development of symptoms in post-traumatic stress disorder (PTSD) is determined by a set of factors, which are not limited to classical neurotransmitter systems in the brain or stress hormones. In particular, the brain renin-angiotensin-aldosterone system may be involved in the mechanisms of PTSD.

Objective. To study the effect of hACE2 expression, angiotensin-converting enzyme 2 (ACE2) gene, on anxiety and susceptibility to psycho-physiological stress in mice in the foot electroshock (FS) model of PTSD.

Materials and methods. The experiments were conducted using 4–5-month-old male C57Bl/6N and k18-hACE2-KI mice. C57Bl/6N mice were divided into three groups: control (n = 7); the foot shock (FS) (n = 7); FS + lisinopril (n = 7). k18-hACE2-KI mice were divided into two groups: control (n = 7) and the FS (n = 8). Pavlovian fear conditioning was performed using FS as an unconditioned stimulus. Mice in the FS + lisinopril group received lisinopril at a dose of 10 mg/kg per day with drinking water for 28 days after psychophysiological trauma. The expression of fear, reflecting the memory of psychophysiological trauma, was assessed on day 7 and day 28 after FS exposure. The magnitude of the fear response was assessed by evaluation of the relative time of freezing. The open field test was used to assess general locomotor activity. The tail suspension test was used to assess the stress-coping strategy, while the light-dark box test and the elevated plus maze test were used to measure anxiety. The Barnes maze test was used to explore spatial navigation and spatial learning dynamics. Behavior was analyzed using the ANY-maze Video-Tracking Software. Statistical analysis was performed using the Prism GraphPad v.10.0 software.

Results. k18-hACE2-KI mice with expression of humanized ACE2 gene under the control of the cytokeratin gene promoter showed a more pronounced ability to remember and retain the memory about the conditioned stimulus/context of the traumatic event in the PTSD-model when compared to C57Bl/6N mice. Anxiety measured in the light-dark box test was lower in k18-hACE2 mice than C57Bl/6N mice after FS. At the same time, there was a decrease in the open-field motor activity and there were no changes in spatial memory in the Barnes maze test. Lisinopril, an ACE inhibitor (28 days after FS), did not reduce traumatic memory in C57Bl/6N mice, indicating that the promnestic effect of hACE2 gene expression is not a result of systemic hypotension and pointing at the involvement of the central mechanisms in the realization of hACE2 gene effect in the pathological phenotype development.

Conclusions. The data indicate that the hACE2 gene affects the stress response in mice. Specifically, the expression of hACE2 gene in mice leads to increased memory of psychophysiological trauma and reduced extinction of traumatic memory compared to wild-type mice. This may be due to the modulation of the ACE2-dependent renin-angiotensin-aldosterone system in the brain. The decreased RAAS activity under the action of the ACE inhibitor lisinopril with a hypotensive effect did not affect memory in wild-type mice.

1. Wang CX, Kohli R, Olaker VR, Terebuh P, Xu R, Kaelber DC, et al. Risk for diagnosis or treatment of mood or anxiety disorders in adults after SARS-CoV-2 infection, 2020–2022. Molecular Psychiatry. 2024;29(5):1350–60. https://doi.org/10.1038/s41380-024-02414-x

2. Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020;181(2):271–80.e8. https://doi.org/10.1016/j.cell.2020.02.052

3. Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne M.A, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 2003;4269(6965):450–4. https://doi.org/10.1038/nature02145

4. Li XC, Zhang J, Zhuo JL. The vasoprotective axes of the renin-angiotensin system: Physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases. Pharmacological Research. 2017;125:21–38. https://doi.org/10.1016/j.phrs.2017.06.005

5. Correa BHM, Becari L, Fontes MAP, Simões-e-Silva AC, Kangussu LM. Involvement of the Renin-Angiotensin System in Stress: State of the Artand Research Perspectives. Current Neuropharmacology. 2022;20(6):1212–28. https://doi.org/10.2174/1570159X19666210719142300

6. Yang XH, Deng W, Tong Z, Liu YX, Zhang LF, Zhu H, et al. Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection. Comparative Medicine. 2007;57(5):450–9.

7. Cheyne I, Gopinath VS, Muppa N, Armas AE, Gil Agurto MS, Akula SA, et al. The Neurological Implications of COVID-19: A Comprehensive Narrative Review. Cureus. 2024;16(5):e60376. https://doi.org/10.7759/cureus.60376

8. Lima AM, Xavier CH, Ferreira AJ, Raizada MK, Wallukat G, Velloso EP, et al. Activation of angiotensin-converting enzyme 2/ angiotensin-(1–7)/Mas axis attenuates the cardiac reactivity to acute emotional stress. American Journal of Physiology: Heart and Circulatory Physiology. 2013;305(7):H1057–67. https://doi.org/10.1152/ajpheart.00433.2013

9. Meng Y, Yu CH, Li W, Li T, Luo W, Huang S, et al. Angiotensin-Converting Enzyme 2/Angiotensin-(1-7)/Mas Axis Protects against Lung Fibrosis by Inhibiting the MAPK/ NF-κB Pathway. American Journal of Respiratory Cell and Molecular Biology. 2014;50(4):723–36. https://doi.org/10.1165/rcmb.2012-0451OC

10. Grobe JL, Xu D, Sigmund CD. An Intracellular Renin-Angiotensin System in Neurons: Fact, Hypothesis, or Fantasy. Physiology. 2008;23(4):187–93. https://doi.org/10.1152/physiol.00002.2008

11. Martinelli S, Anderzhanova EA, Bajaj T, Wiechmann S, Dethloff F, Weckmann K, et al. Stress-primed secretory autophagy promotes extracellular BDNF maturation by enhancing MMP9 secretion. Nature Communication. 2021;12(1):4643. https://doi.org/10.1038/s41467-021-24810-5

12. Azaryan A, Barkhudaryan N, Galoyan A, Lajtha A. Action of brain cathepsin B, cathepsin D, and high-molecular-weight aspartic proteinase on angiotensins I and II. Neurochemical Research. 1985;10(11):1525–32. https://doi.org/10.1007/BF02430602

13. Rukavina Mikusic NL, Pineda AM, Gironacci MM. Angiotensin-(1-7) and Mas receptor in the brain. Exploration of Medicine. 2021;2(3):268–93. https://doi.org/10.37349/emed.2021.00046

14. Kerr DS, Bevilaqua LRM, Bonini JS, Rossato JI, Köhler CA, Medina JH, et al. Angiotensin II blocks memory consolidation through an AT2 receptor-dependent mechanism. Psychopharmacology. 2005;179(3):529–35. https://doi.org/10.1007/s00213-004-2074-5

15. De Kloet AD, Cahill KM, Scott KA, Krause EG. Overexpression of angiotensin converting enzyme 2 reduces anxiety-like behavior in female mice. Physiology & Behavior. 2020;224:113002. https://doi.org/10.1016/j.physbeh.2020.113002

16. Wang L, De Kloet AD, Pati D, Hiller H, Smith JA, Pioquinto DJ, et al. Increasing brain angiotensin converting enzyme 2 activity decreases anxiety-like behavior in male mice by activating central Mas receptors. Neuropharmacology. 2016;105:114–23. https://doi.org/10.1016/j.neuropharm.2015.12.026

17. Kao CY, Stalla G, Stalla J, Wotjak CT, Anderzhanova E. Norepinephrine and corticosterone in the medial prefrontal cortex and hippocampus predict PTSD-like symptoms in mice. European Journal of Neuroscience. 2015;41(9):1139–48. https://doi.org/10.1111/ejn.12860

18. Kamprath K, Wotjak CT. Nonassociative learning processes determine expression and extinction of conditioned fear in mice. Learning Memory. 2004;11(6):770–86. https://doi.org/10.1101/lm.86104

19. Motulsky HJ, Brown RE. Detecting outliers when fitting data with nonlinear regression — a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics. 2006;7(1):123. https://doi.org/10.1186/1471-2105-7-123

20. Yamagata R, Nemoto W, Nakagawasai O, Takahashi K, Tan-No K. Downregulation of spinal angiotensin converting enzyme 2 is involved in neuropathic pain associated with type 2 diabetes mellitus in mice. Biochemical Pharmacology. 2020;174:113825. https://doi.org/10.1016/j.bcp.2020.113825

21. Albrecht D. Angiotensin-(1-7)-induced plasticity changes in the lateral amygdala are mediated by COX-2 and NO. Learning Memory. 2007;14(3):177–84. https://doi.org/10.1101/lm.425907

22. Hellner K, Walther T, Schubert M, Albrecht D. Angiotensin-(1-7) enhances LTP in the hippocampus through the G-protein-coupled receptor Mas. Molecular and Cellular Neuroscience. 2005;29(3):427–35. https://doi.org/10.1016/j.mcn.2005.03.012

23. Almeida-Santos AF, Kangussu LM, Moreira FA, Santos RAS, Aguiar DC, Campagnole-Santos MJ. Anxiolytic- and antidepressant-like effects of angiotensin-(1–7) in hypertensive transgenic (mRen2)27 rats. Clinical Science. 2016;130(14):1247–55. https://doi.org/10.1042/CS20160116

24. Kangussu LM, Almeida-Santos AF, Bader M, Alenina N, Fontes MA, Santos RA, et al. Angiotensin-(1-7) attenuates the anxiety and depression-like behaviors in transgenic rats with low brain angiotensinogen. Behavioural Brain Research. 2013;257:25–30. https://doi.org/10.1016/j.bbr.2013.09.003

25. Fontes MA, Martins Lima A, dos Santos RAS. Brain angiotensin-(1-7)/Mas axis: A new target to reduce the cardiovascular risk to emotional stress. Neuropeptides. 2016;56:9–17. https://doi.org/10.1016/j.npep.2015.10.003

26. Marvar PJ, Goodman J, Fuchs S, Choi DC, Banerjee S, Ressler KJ. Angiotensin Type 1 Receptor Inhibition Enhances the Extinction of Fear Memory. Biological Psychiatry. 2014;75(11):864–72. https://doi.org/10.1016/j.biopsych.2013.08.024

27. Braszko JJ. AT 2 but not AT 1 receptor antagonism abolishes angiotensin II increase of the acquisition of conditioned avoidance responses in rats. Behavioural Brain Research. 2002;131(1–2):79–86. https://doi.org/10.1016/S0166-4328(01)00349-7

28. Raghavendra V, Chopra K, Kulkarni SK. Comparative studies on the memory-enhancing actions of captopril and losartan in mice using inhibitory shock avoidance paradigm. Neuropeptides. 2001;35(1):65–9. https://doi.org/10.1054/npep.2000.0845

29. Cohen H, Kaplan Z, Koresh O, Matar MA, Geva AB, Zohar J. Early post-stressor intervention with propranolol is ineffective in preventing posttraumatic stress responses in an animal model for PTSD. European Neuropsychopharmacology. 2011;21(3):230–40. https://doi.org/10.1016/j.euroneuro.2010.11.011

30. Nylocks KM, Michopoulos V, Rothbaum AO, Almli L, Gillespie CF, Wingo A, et al. An angiotensin-converting enzyme (ACE) polymorphism may mitigate the effects of angiotensin-pathway medications on posttraumatic stress symptoms. American Journal of Medical Genetics Part B. 2015;168(4):307–15. https://doi.org/10.1002/ajmg.b.32313

31. Ferrario CM, Jessup J, Chappell MC, Averill DB, Brosnihan KB, Tallant EA, et al. Effect of Angiotensin-Converting Enzyme Inhibition and Angiotensin II Receptor Blockers on Cardiac Angiotensin-Converting Enzyme 2. Circulation. 2005;111(20):2605–10. https://doi.org/10.1161/CIRCULATIONAHA.104.510461

32. Imai Y, Kuba K, Rao S, Huan Y, Guo F, Guan B, et al. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature. 2005;436(7047):112–6. https://doi.org/10.1038/nature03712

33. Ishiyama Y, Gallagher PE, Averill DB, Tallant EA, Brosnihan KB, Ferrario CM. Upregulation of Angiotensin-Converting Enzyme 2 After Myocardial Infarction by Blockade of Angiotensin II Receptors. Hypertension. 2004;43(5):970–6. https://doi.org/10.1161/01.HYP.0000124667.34652.1a

34. Karram T, Abbasi A, Keidar S, Golomb E, Hochberg I, Winaver J, et al. Effects of spironolactone and eprosartan on cardiac remodeling and angiotensin-converting enzyme isoforms in rats with experimental heart failure. American Journal of Physiology: Heart and Circulatory Physiology. 2005;289(4):H1351–8. https://doi.org/10.1152/ajpheart.01186.2004

35. Supé S, Kohse F, Gembardt F, Kuebler WM, Walther T. Therapeutic time window for angiotensin-(1–7) in acute lung injury. British Journal of Pharmacology. 2016;173(10):1618–28. https://doi.org/10.1111/bph.13462